Olfactory Progenitor Cell Transplantation

Into The Mammalian Inner Ear

Nirmal Praful Patel M.B.B.S. (Hons), F.R.A.C.S. (ORLHNS)

A thesis submitted in fulfillment of the requirements for a

Master of Surgery by Research Thesis

Enrolled at the

Garvan Institute of Medical Research

Faculty of Medicine

The University of New South Wales

Work conducted at the

Garvan Institute of Medical Research and the

Laboratory of Molecular Otology,

New York University, New York, New York, USA

Resubmitted August 2006

2

Dedication

To my wife Yuvisthi,

The initiator, guiding influence and constant support,

Thank you.

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Abbreviations

AraC Arabinocytosine

BDNF brain-derived neurotrophic factor

BSA bovine serum albumin

cDNA complementary deoxyribonucleic acid

CNS central nervous system

DMEM Dulbecco’s Modified Eagle’s Medium

DNAse deoxyribonuclease

DRG Dorsal Root Ganglion

E embryonic day

EDTA disodium ethylenediaminetetracetic acid

EGF epidermal growth factor

ES embryonic stem cells

FACS fluorescence-activated cell sorting

FBS foetal bovine serum

FGF-2 fibroblast growth factor 2

GDNF glial cell line-derived neurotrophic factor

GFAP glial fibrillary acidic protein

GFP green fluorescent protein

HEPES N-2-hydroxyethylpiperazine-N’-2-ethanesulphonic acid

MEM D-Val Minimum Essential Medium D-Valine

NGS normal goat serum

4

NHS normal horse serum

NSC neural stem cell

NT3 Neurotrophin 3

ON olfactory neuroepithelium

ONS Olfactory Neurosphere

OPC olfactory progenitor cell

P Postnatal

PBS phosphate buffered saline

PFA Paraformaldehyde

RT room temperature

SGN Spiral Ganglion Neuron

SM Scala Media

ST Scala Tympani

SV Scala Vestibuli

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Acknowledgements

Undertaking research in this field has been both a significant challenge and a

gradual joy. No such work is accomplished alone. To the following people I owe many

thanks.

Mona Khan for her support and guidance in my initial endeavours in science.

Peter Schofield for encouraging, supporting and guiding me through this whole project.

Anil Lalwani and Anand Mhatre for spending much time teaching me to think

scientifically and write with clarity. Clough Shelton for improving my critical thinking

and attention to detail. The Garnett Passe Rodney Williams Foundation for providing

encouragement to a recently graduated otolaryngologist with little scientific track record,

who wanted to try and think with more rigour. Dr John Tonkin for supporting my

application to the Sisters of Charity. The National Organization for Hearing Research for

identifying and supporting my “high risk” project. To my family and especially Mum,

Dad, Mamasa and Boyapa, who continue to teach and show me the meaning of

generosity. Lastly and most importantly, to Yuvisthi, Shyaan and Zuvhir; thank you for

enduring my absence with little complaint and continuous love.

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Preface

This thesis was based on the following manuscripts:

Biological Therapy for the Inner Ear

Patel, N.P., Mhatre, A.N. and Lalwani, A.K.

(Expert Opin Biol Ther, Nov 2004: 4(11):1811-9)

Factors Effecting The Survival Of Neural Stem Cells In Inner Ear Explants

Patel, N.P., Mhatre, A.N. and Lalwani, A.K.

(Submitted to Hearing Research December 2005)

Olfactory Neural Progenitor Cell Transfer Into The Mammalian Cochlea

Patel, N.P., Mhatre, A.N. and Lalwani, A.K.

(Submitted to Hearing Research December 2005)

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Table of Contents Chapter 1 Introduction .................................................................................................10

1.1 Biological Therapy Of The Inner Ear .................................................................................10

1.2 Early Inner Ear Development...............................................................................................11

1.3 Hair Cell Regeneration ..........................................................................................................15

1.4 Gene Therapy of the Inner Ear ............................................................................................17 1.4.1 Viral Vectors in Intra-Cochlea Gene Therapy ................................................................................18 1.4.2 Non Viral Vectors for Intra-Cochlea Gene Transfers.....................................................................19 1.4.3 Transgene Expression .......................................................................................................................22

1.5 Suitable Animal Models for Biological Therapy of the Inner Ear..................................22

1.6 Delivery Modalities for Biological Therapy of the Inner Ear..........................................24

1.7 Pre-clinical Applications of Gene Therapy.........................................................................27

1.8 Cellular Therapy of the Inner Ear .......................................................................................32 1.8.1 Graft Sources for Cellular Therapy in the Inner Ear ......................................................................32

1.8.2.1 Stem Cells .................................................................................................................................33 1.8.2.2 Neural Stem Cells (NSC) .........................................................................................................33

1.8.3 Successful Cellular Grafts in the Inner Ear .....................................................................................34 1.8.4 Neural Stem Cells for Cellular Therapy of the Inner Ear...............................................................39 1.8.5 Neural Stem Cells as Gene Therapy Vectors ..................................................................................40

1.9 Risks and Limitations of Intra-cochlea Biological Therapy ............................................41

1.10 Neurobiology of the Olfactory Epithelium .......................................................................43 1.10.1 The Olfactory Epithelium...............................................................................................................43 1.10.2 Olfactory Progenitor Cells .............................................................................................................44 1.10.3 Olfactory Progenitor Cells Grown in Vitro...................................................................................46 1.10.4 Nestin in Olfactory Epithelium and Olfactory Spheres................................................................46

1.11 Aims of This Thesis...............................................................................................................47

Chapter 2 Materials and Methods ................................................................................49

2.1 Animal Surgical Methods ......................................................................................................49 2.1.1 Harvesting and Preparation of the Cochleovestibular Explant ......................................................49

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2.1.2 Stem Cell Introduction into In vitro Models ...................................................................................50 2.1.3 Olfactory Epithelium Harvesting .....................................................................................................54 2.1.4 GFP OPC Introduction into the In Vivo Adult Mouse Model........................................................55

2.2 Cell Culture Methods .............................................................................................................56 2.2.1 NSC C17.2 – Growth and Preparation Assays................................................................................56 2.2.2 Olfactory Neurosphere Isolation and Culture .................................................................................58

2.2.2.1 Respiratory and Olfactory Tissue Size Fractionation.............................................................58 2.2.2.2 Passaging and Nestin Staining of Olfactory Spheres .............................................................59 2.2.2.3 Differentiating Olfactory Spheres............................................................................................60 2.2.2.4 Preparing Olfactory Neurospheres for Microinjection...........................................................60

2.3 Fixation Methods ....................................................................................................................61

2.4 Staining and Immunohistochemical Methods ....................................................................62

2.5 Microscopy and Digital Imaging Methods..........................................................................63

2.6 Statistical Methods..................................................................................................................64

Chapter 3 an In vitro Model For Cellular Therapy of The Inner Ear..........................66

3.1 Introduction .............................................................................................................................66

3.2 Results .......................................................................................................................................68 3.2.1 Cochleovestibular Explants Survive Up to 6.25 Days ...................................................................68 3.2.2 Microinjection Is a More Effective Method of Stem Cell Delivery into the Explants.................68 3.2.3 C17.2 Cells Are Optimised for Delivery at a Confluence of 70% & Concentrations of 1 Million

Cells / mL....................................................................................................................................................72 3.2.4 Ototoxins Damage the Organ of Corti in Explants .........................................................................76 3.2.6 Brain Derived Nerve Growth Factor (BDNF) Improves C17.2 Cell Survival .............................84

3.3 Discussion .................................................................................................................................84

Chapter 4 Olfactory Progenitor Cell Transplantation into Mammalian Inner Ears ....91

4.1 Introduction .............................................................................................................................91

4.2 Results .......................................................................................................................................93 4.2.1 Olfactory Spheres with Nestin Positive Cells Can Be Isolated from the Mouse Olfactory

Epithelium...................................................................................................................................................93 4.2.3 Differentiation of Olfactory Spheres into ß tubulin and GFAP Positive Cells .............................95 4.2.4 Olfactory Neurosphere and Olfactory Progenitor Derived Cells Survived Poorly in

Cochleovestibular Explants .......................................................................................................................96

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4.2.3 ONS and OPC Derived Cells Survive Robustly In Vivo ..............................................................100 4.2.4 Microinjection of the In Vivo Cochlea Caused No Structural Damage and No Injected Cells

Were Found in Contralateral Inner Ears .................................................................................................108

4.3 Discussion ...............................................................................................................................108

Chapter 5 General Discussion ....................................................................................115

5.1 Core Issues in the Development of Inner Ear Biological Therapy................................115

5.2 Research Directions for Future Work...............................................................................117

5.3 Concluding Remarks ............................................................................................................121

Chapter 6 References..................................................................................................122

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Chapter 1 Introduction

1.1 Biological Therapy Of The Inner Ear

Deafness in either a partial or complete form affects 13.5% of Australians (Australian

Bureau of Statistics 2001). The vast majority, up to 90%, of hearing loss is sensorineural in

aetiology. The management of sensorineural hearing loss currently relies upon electrical

rehabilitation in the form of hearing aids and cochlea implantation. Although this method is

effective in creating serviceable hearing in the appropriately selected patient, it does little to

repair or regenerate the natural physiological mechanisms of mechanical energy effectively

transduced into electrical energy that are required for interpretation of acoustically complex

signals such as music. Biological therapies, in the form of either hair cell or spiral ganglion

repair are attractive in that they may potentially recreate this normal, highly efficient,

physiological scenario.

To date, biological therapy of inner ear pathology is in its infancy. Following the

demonstration of avian inner ear hair cell regeneration after injury, considerable progress

has been made towards the molecular understanding of this mechanism (Corwin and

Cotanche 1988; Cruz et al. 1987; Ryals and Rubel 1988). Spontaneous hair cell

regeneration in the cochlea has been demonstrated in lower vertebrate animals, but not in

mammals. Ultrastructural evidence supporting spontaneous regeneration of mammalian

vestibular hair cells following ototoxic insult does exist (Forge et al. 1993; Warchol et al.

1993). More recently, mature mammalian vestibular epithelium has been demonstrated

to contain a small number of regenerating stem cells (Li et al. 2003a). However, the

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presence of a similar subpopulation of regenerating cells has not been observed in the

adult mammalian organ of Corti.

1.2 Early Inner Ear Development

An understanding of the molecular mechanisms of inner ear development

provides a rationale for both understanding hair cell/ spiral ganglion regeneration and for

purposefully applying biological therapy to the region. The mammalian inner ear

develops from a neuroectodermal thickening called the otic placode. Invagination of

each placode into a pit and subsequently an otocyst, is the prelude to membranous

labyrinth formation. The ventromedial region of each otic cyst is delineated as the

prosensory region. This region ultimately forms the six discrete sense organs of the inner

ear; namely the three cristae of the semicircular canals, two maculae of the labyrinth and

the cochlea’s organ of Corti. The remainder of the otic cyst forms the chambers of the

membranous labyrinth.

The formation of the sensory organs is dependant upon the Notch receptor

pathway. Notch is a cell membrane receptor distributed widely in mammalian neural

development and critical in glial cell differentiation in the CNS (Ahmad et al. 1995; Del

Amo et al. 1992; Higuchi et al. 1995; Weinmaster et al. 1991). Activation of this

receptor by its various ligands including Delta1, Jagged 1/ 2 and Numb is essential for

sensory organ growth. Notch activation allows growth of the sense organ patch and

prevents premature hair cell differentiation (Bober et al. 2003; Bryant et al. 2002;

Lanford et al. 1999; Rinkwitz et al. 2001). Each sensory organ probably develops into its

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unique area by the asymmetric overlapping expression of particular genes, of which, only

a handful are currently known.

Sensory organs are at present thought to develop from a single patch of sensory

competence in the developing embryo. This sensory area under the influence of different

temporally and spatially acting genes defines unique sites (Montcouquiol and Kelley

2003). Two proteins first widely label the primordial auditory and vestibular sensory

area. Bone Morphogenic Protein 4 (BMP4) and Lunatic fringe (fng) mark out discrete

areas in the ventromedial otocyst (Cole et al. 2000; Morsli et al. 1998; Zhang et al. 2000).

FGFR10 is also expressed in early formation (Pauley et al. 2003). The presence of these

three proteins along with transcription factors such as Pax2, Otx1 and D1x5, appears to

crudely delineate the formation of particular inner ear sense areas (Oesterle and Hume

1999). Sequentially with sensory patch specification, mechanoreceptor hair cells and

their supporting cells develop.

Hair cells and supporting cells are derived from a common cell lineage.

Differentiation of each cell type most likely occurs by process of cell differentiation from

a single homogenous population, known as lateral inhibition. Again, Notch receptor

activation is critical for lateral inhibition (Lanford et al. 1999). This mechanism, along

with coordinated transcription factor activation directs hair and supporting cell formation.

The mechanism varies with animal models, however, knockout technology is enabling

better definition of the developing mammalian cochlea hair cell. Math1 (the gene for a

basic helix loop helix transcription factor) expression is critical for a hair cell to form

once it has left the cell cycle (Fritzsch et al. 2002; Woods et al. 2004; Zheng and Gao

2000). Following Math 1 expression, Notch receptor inhibition occurs (via ligands Delta

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1, Jagged 1 and 2) to stimulate hair cell formation and laterally inhibit adjacent cells to

undergoing the same fate (Lanford et al. 1999; Morrison et al. 1999). Lastly, Brain 3c (a

POU domain transcription factor) appears important in the final stages of hair cell

differentiation (Xiang et al. 1997a; Xiang et al. 1997b; Xiang et al. 1998). Supporting

cells, in contrast, require elevated levels of Notch stimulation to prevent them becoming

hair cells (Bryant et al. 2002).

Cochleovestibular ganglia neurons are derived from the otic placode and inner ear

innervation is precise and tonotopic (Rubel and Fritzsch 2002). All sensory neurons of

the ear require Neurogenin 1 (a basic helix loop helix proneural gene) for their formation

(Ma et al. 2000). The growth and migration of developing neurons from the brain stem to

innervate the ear is not well understood. Neurons projecting from the brainstem to the

inner ear probably have an identity that directs them to their target. One distinct marker –

the zinc finger protein GATA enhancer binding protein 3 (GATA3) – is well established

in the developing spiral ganglion. GATA3 is probably involved in routing developing

fibres (Lawoko-Kerali et al. 2002). Innervation of the developing ear to the brain is also

critically dependent upon neurotrophins.

Neurotrophins are a group of soluble growth factor polypeptides that share 60%

amino acid homology and serve key roles in neuronal development and survival

throughout the peripheral and central nervous system. Mammalian neurotrophins are

generally divided into four groups - Brain Derived Neurotrophic Factor (BDNF), Nerve

Growth Factor (NGF), Neurotrophin 3 (NT3), Neurotrophin 4/ 5 (NT4/5). Neurotrophin

proteins act via a high affinity specific ligand/ receptor interaction with Trk family of

tyrosine kinase receptors. Three Trk receptors exist – Trk A, B and C (Despres and

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Romand 1994; Staecker et al. 1996). Each receptor binds a specific ligaND: Trk A binds

NGF, Trk C binds NT3 and Trk B interacts with BDNF and NT4/5 (Parada et al. 1992;

Tessarollo et al. 1993). Binding of the individual neurotrophins to the Trk receptor

triggers an intrinsic tyrosine kinase activity resulting in autophosphorylation of tyrosine

residues. Neurotrophic action ultimately occurs via various intracellular molecules (ras,

PI-3 kinase and Phospholipase C-1) that bind to these tyrosine kinase residues and

activate intracellular cascades, ultimately effecting neurotrophic actions (Barker and

Murphy 1992; Schecterson and Bothwell 1992). In addition, neurotrophins bind to a

transmembrane glycoprotein, p75 low affinity growth factor receptor in the cochlea, the

outcome of this interaction is not known (Knipper et al. 1999; Schecterson and Bothwell

1994).

Two neurotrophins are particularly important in inner ear afferent and efferent

innervation, BDNF and NT3. Knockout studies have demonstrated the crude

topotonicity of the neurotrophic effect of these two neurotrophins suggesting they play a

major role in neuronal migration and synaptogenesis (Despres and Romand 1994;

Fritzsch et al. 2002; Fritzsch et al. 1997c; Malgrange et al. 1996; Mou et al. 1997;

Schecterson and Bothwell 1994; Wheeler et al. 1994; Zheng et al. 1995). Brain derived

neurotrophic factor (BDNF) is critical for vestibular ganglion formation and vestibular

innervation. Mouse knockouts of BDNF and its receptor TrkB lack innervation to all hair

cells of the semicircular canals, utricle and saccule (Fritzsch et al. 1997b; Fritzsch et al.

1997c). Furthermore, these mice lack innervation to the apical turns of the cochlea. Mice

lacking NT3 and its receptor TrkC lack innervation to the basal turns of the cochlea

15

(Ernfors et al. 1995; Fritzsch et al. 1997b). BDNF and NT3 mutants lack any inner ear

innervation (Fritzsch et al. 1999).

1.3 Hair Cell Regeneration

As recently as 20 years ago it was generally accepted that all warm blooded animals

had a complete set of hair cells at birth and any subsequent damage to these cells was

irreversible. In 1987 and 1988 reports of avian hair cell regeneration were first published

demonstrating light and scanning electron microscopic evidence of hair cell regeneration

following both acoustic and ototoxic damage (Corwin and Cotanche 1988; Cotanche 1987a;

Cotanche 1987b; Cruz et al. 1987; Ryals and Rubel 1988). Since then considerable evidence

has also accumulated that these morphologically recovering hair cells are in fact

electrophysiologically and functionally recovering as well (Reng et al. 2001; Woolley and

Rubel 2002; Woolley et al. 2001). Although the evidence for functional audiological

recovery in birds following hair cell damage is considerable, the completeness and duration

of this recovery has not been validated (Muller and Smolders 1999). Spontaneous hair cell

regeneration in the mammalian cochlea following injury has not been demonstrated to date,

although there is evidence for hair cell regeneration in the mammalian vestibule (Forge et al.

1993; Kirkegaard and Jorgensen 2000; Warchol et al. 1993) and following exogenous gene

transfer in the mammalian cochlea (Kawamoto et al. 2003).

Current work in both the avian and mammalian models of hair cell regeneration

emphasizes the importance of the supporting cell in the organ of Corti (or its avian equivalent

– the basilar papilla). Conceptually four possible mechanisms exist for hair cell regeneration

16

in the cochlea. The first two mechanisms directly involve the supporting cell. Firstly,

generation of new hair cells could be achieved by mitosis of supporting cells, with progeny

ultimately differentiating into mature hair cells and replacement supporting cells (Raphael

1992; Raphael 1993; Raphael et al. 1994; Stone and Rubel 2000). Secondly, there is

evidence that supporting cells undergo phenotypic conversion to hair cells without mitosis, a

process referred to as transdifferentiation (Adler and Raphael 1996; Kawamoto et al. 2003;

Roberson and Rubel 1994) . Recently immunophenotypic evidence has been published,

identifying around 30% of regenerated hair cells in the avian model that bypass mitosis and

undergo phenotypic conversion from supporting cells to hair cells (Morest and Cotanche

2004; Roberson et al. 2004). Thirdly, an unidentified stem cell may reside in the organ of

Corti and be coaxed toward mitosis, the end product of which could be a mature hair cell.

The rationale for this presupposition in the organ of Corti hair cells, is that the mammalian

vestibular epithelium appears to have a retained capacity for self renewal and regeneration,

with the demonstration of stem cells existing in utricular epithelium by Li and colleagues (Li

et al. 2003a). Lastly, transplanted cells could be introduced and ultimately coaxed toward a

hair cell fate.

The supporting cell in the mammalian organ of Corti was until recently, thought to

undergo terminal mitosis during embryogenesis. There is some evidence for non-sensory

epithelium to undergo regeneration and form at least, partially functioning hair cells via over

expression of the key hair cell development gene Math 1 (Izumikawa et al. 2005). Since

Math 1 over expression is unlikely to cause mitosis (Zheng and Gao 2000), it was inferred

that this was either a transdifferentiation phenomenon involving the supporting cells or

17

alternatively was a coaxing of as yet unidentified stem cells in the organ of Corti toward

developing new hair cells produced the phenomenon of hair cell growth.

Since hair cell loss is the major outcome of most forms of sensorineural hearing

loss, their regeneration and repair through application of cellular and molecular therapies

represents a major focus of current hearing research. Two broad areas of biological

therapy have evolved in the last 20 years – gene and cellular therapy.

1.4 Gene Therapy of the Inner Ear

The simple and powerful objective of gene transfer technology is to introduce a

‘therapeutic gene’, for example a normal version of the defective gene, into appropriate target

cells of the affected individual. Expression of the exogenous ‘therapeutic gene’ would then

alter the target cell and the clinical phenotype.

Both viral and non-viral vectors have been used to transfer and express genes in the

inner ear of animal models, however, non-viral vectors are rather inefficient and await further

development. Individual virions represent a highly efficient means of introducing the viral

genome into the nuclei of target cells followed by the use of the cellular machinery to express

the viral genes. These viral agents have been adapted for the purpose of gene transfer by

altering their genome so that they can no longer replicate within the transduced cell and lead

to cellular lysis. These replication defective viruses are engineered to function solely to

introduce the desired gene into the nuclei of target cells. Viral vectors have been developed

from both DNA (e.g., adeno, adeno-associated and herpes) and RNA (e.g., retro and

influenza) viruses.

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1.4.1 Viral Vectors in Intra-Cochlea Gene Therapy

Feasibility of gene therapy for inner ear pathology was initially demonstrated

through intra-cochlea transgene expression using the guinea pig as an animal model

(Lalwani et al. 1996). A viral vector derived from the adeno associated virus (AAV)

capable of transducing non-dividing cells and considered to be safer than other viral

vectors, in view of its non-immunogenicity and non-pathogenicity, was used to deliver a

marker transgene to the cochlea via steady state infusion using an osmotic minipump.

Based upon expression of the marker transgene encoding a bacterial enzyme β-

galactosidase, AAV vector was found to transduce the spiral limbus, spiral ligament,

spiral ganglion and the organ of Corti (Lalwani et al. 1996). The marker gene expression

was shown to be present up to 24 weeks after osmotic minipump mediated infusion of the

AAV-βgal (Lalwani et al. 1996). Recent work using AAV and modifying the promoter

driving the marker gene from chicken beta actin/ cytomegalovirus promoter to GFAP

promoter has succeeded in selectively expressing the marker transgene in supporting cells

of the inner ear (Stone et al. 2005). Subsequent studies investigating intra-cochlea gene

transfer have characterized a variety of different vectors for their efficacy and safety as

well as their mode of introduction into the cochlea.

The adenoviral (Ad) vector represents one of the well-characterized viral vectors

used for intra-cochlea gene transfer. The attributes of adenoviral vector for gene transfer

include its capacity to carry large transgenes (8 kb), it can be generated at high titre and it

can transduce both dividing and non-dividing cells. The major disadvantage associated

19

with the use of first generation Ad vector was the strong immune response that it elicited

(Staecker et al. 2001). Subsequent development of adenoviral vectors has generated

attenuated viruses with complete deletion of viral protein sequences, leaving a vector

with all the advantages and diminishing, if not eliminating, its immunogenicity

(Amalfitano and Parks 2002). This newer generation of adenovirus still awaits testing in

the inner ear environment.

A lentiviral vector, based on the human immunodeficiency virus (HIV), can

integrate into the chromosome of both dividing and non-dividing or mitotically quiescent

cells leading to a potentially stable, long-term expression of a transgene spliced into the

viral vector. Thus, the post-mitotic cochlea neuroepithelia and the spiral ganglion

neurons represent suitable targets for a stable long-term transgene expression via

lentivirus mediated gene transfer. Infusion of the lentiviral vector carrying a marker

transgene into a guinea pig cochlea has revealed highly restricted fluorescence pattern

limited to the periphery of the perilymphatic space (Han et al. 1999). Transduction of

SGN and glial cells by lentivirus in vitro but not in vivo suggests limited dissemination of

the viral vector from the perilymphatic space. Restricted transduction of cell-types

confined to the periphery of the perilymphatic space by the lentivirus is ideal for stable

production of gene products secreted into the perilymph.

1.4.2 Non Viral Vectors for Intra-Cochlea Gene Transfers

In addition to viral vectors, cationic lipid vesicles or liposomes have also been used

for intra-cochlea gene transfer (Staecker et al. 2001; Wareing et al. 1999). The liposomes

20

coupled to the transgene integrated within a plasmid vector, binds to the plasma membrane of

the target cells, releasing the DNA into the cytoplasm where it is eventually incorporated into

the host genome. Liposome vectors are non-immunogenic and are easy to produce.

Furthermore, the DNA introduced into the host cell is incorporated by recombination, so

there is little risk of insertional mutagenesis. The drawback of liposome vectors is a low

transfection rate compared with other vectors. The feasibility of inner ear gene transfer with

liposome vectors has been demonstrated (Wareing et al. 1999). The attributes of the gene

transfer vectors that have been used in intra-cochlea gene transfer are summarized in Table 1

(see page 21). These parameters provide a guide for assessing suitability of a given vector

for specific objective. Thus, if a transgene expression is required for a short period only,

then adenoviral vector or liposomes may be suitable. However, if sustained gene expression

is required that replaces a non-functional mutant gene product, then a retroviral vector or

adeno-associated virus would be more suitable as expression of the transgene is dependant

upon insertion of the viral DNA into the host cell genome.

21

Table 1. Characteristics of Gene Transfer Vectors Used in the Inner Ear

VECTOR Genome Insert Size Site Efficiency Cell Division Expression Advantages Disadvantages

AAV ssDNA 4.5 kB Genome Variable Not required Permanent No human disease

Difficult to produce

Retrovirus RNA 6 - 7 kB Genome Low Required Permanent Suited for neoplastic

cells

Insertional mutagenesis

Adenovirus dsDNA 7.5 kB Episome Moderate Not required Transient Ease of production

Inflammatory response

Herpes virus

dsDNA 10-100 kB Episome Moderate Not required Transient Neural tropism

Human disease

Plasmid RNA/ DNA

Unlimited Episome Very low Not required Transient Safe, Easy Production

Low transfection

Liposome RNA/ DNA

Unlimited Episome Very low Not required Transient Safe, Easy Production

Low transfection

AAV; Adenoassociated Virus (Lalwani AK et al 2002, Patel NP et al 2005,)

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1.4.3 Transgene Expression

The different cell types/tissues transduced by various expression vectors most likely

reflect the unique properties of individual transfer vectors, each being similarly introduced

within the cochlea perilymph carrying marker genes driven by strong viral promoters (Table

2 – see page 23). The variability in the transgene expression pattern is also a consequence of

a number of factors including the size of the viral particle, presence or absence of viral

receptors and mode of delivery, among others. A generalization about the ability of various

viral vectors in transfecting cochlea tissues is that the spiral ganglion cells, spiral ligament

and Reissner’s membrane were transfected by every virus tested. On the other hand, only

adenovirus demonstrated transgene expression within the stria vascularis. Immune response

was present in the cochlea following transfection with adenovirus, HSV and Vaccinia virus.

1.5 Suitable Animal Models for Biological Therapy of the Inner Ear

Preliminary studies in intra-cochlea gene and cell transfer used the guinea pig as the

animal model due to the relatively large size of its cochlea compared to mice and rats and the

ease of surgical manipulation in this species. Subsequent studies are shifting their focus

toward the mouse as the preferred model. The mouse genome is the most extensively

23

Table 2: Transfection of Cochlea Cells and Tissues by Viral Vectors

Supporting Auditory Stria Reissner's Spiral Immune Vector Hair Cells Cells Neurons Vascularis Membrane Ligament Response

AAV + + + - + + - Adenovirus + + + + + + +

Herpes virus - + + - + + + Vaccinia + + - - + + + Lentivirus - - + - + + - Liposomes + + + - + + -

Table 2 represents the various tissues of the cochlea that are transfected by different viral types. The rows contain the outcomes of various viral vectors and where they transfect the tissues. The columns represent the various tissue types. AAV; Adeno Associated Virus. (Lalwani AK et al 2002, Patel NP et al 2004)

24

characterised of all mammalian model organisms. The intrinsic value of the mouse as a

model in hearing research is seen in the availability of a number of mutant mice with

inherited hearing loss (Avraham 2003). These mutant mice have been well characterized and

the genetic basis of their hearing loss identified. In addition, a number of transgenic mice

have also been generated to assess the effect of specific candidate genes whose mutant alleles

have been linked to non-syndromic and syndromic hearing loss (Anagnostopoulos 2002).

Furthermore, established in vitro and in vivo rodent models for ototoxic sensorineural

damage to the inner ear exist.

1.6 Delivery Modalities for Biological Therapy of the Inner Ear

Local biological transfer to the inner ear is feasible because of its relatively closed

anatomy. However, developing a delivery method for genetic vectors/ cells to the inner ear

without causing local destruction and concomitant hearing loss is a significant obstacle. The

general strategy behind these delivery modalities is to introduce the transgene carrying vector

into the inner ear fluid enabling its diffusion to the surrounding tissues. Most of the delivery

methods introduce the gene into the perilymphatic fluid. These methods include

microinjection via the round window membrane, microinjection, or mini-osmotic pump

infusion following cochleostomy and diffusion across the round window membrane after

local Gelfoam placement. Gene transfer vectors have also been introduced into the

endolymphatic fluid through injection into the endolymphatic sac (Yamasoba et al. 1999b) or

into the scala media following cochleostomy (Ishimoto et al. 2002).

25

Histologically, introduction of viral vectors with a mini-osmotic pump was

characterized by an inflammatory response and connective tissue deposition at the basal turn

adjacent to the cochleostomy site. Preservation of pre-operative auditory brain stem response

thresholds in the lower frequencies (1-2 kHz), mild post-operative elevation of thresholds

(<10 dB) in the mid frequencies (4-8 kHz) and marked rise (>30 dB) in auditory brain stem

response thresholds at higher frequencies (>16 kHz) after mini-osmotic pump infusion via a

cochleostomy was also demonstrated (Carvalho and Lalwani 1999). Systemic dexamethasone

-induced immunosuppression has also been shown to largely protect the inoculated ear from

threshold shift and appears to improve adenoviral vector based gene expression (Ishimoto et

al. 2003). Demonstrating ear protection and improved expression with local application of

steroids would clearly be a more optimal situation; limiting the well known systemic side

effects of steroid administration. Presently, however, cochleostomy has been shown to cause

histopathological alterations (including localized surgical trauma and inflammation) and may

lead to hearing loss. In addition, the half-life of the viability of the viral vectors at 37 0C may

also limit or diminish the advantage gained by the sustained and prolonged infusion mediated

by the osmotic mini-pumps.

A much less traumatic alteration to cochleostomy is the direct microinjection through

the round window membrane. Histologically, cochleae microinjected through the round

window demonstrated intact cochlea cytoarchitecture and an absence of inflammatory

response 2 weeks after microinjection via the round window. Further, microinjection

through the round window membrane did not cause permanent hearing dysfunction

(Kawamoto et al. 2001; Stover et al. 2000).

26

To avoid potential hearing loss associated with the direct manipulation of the cochlea,

gene transfer vectors and stem cells have also been delivered through the vestibular apparatus

via canalostomy. This delivery modality yielded transgene expression mainly in the

perilymphatic space with the preservation of cochlea function. Successful delivery of

progenitor cells into the mouse cochlea via the lateral semicircular canal and a cochleostomy

has been achieved and produced little histological change in the mice (Iguchi et al. 2004).

Elevated auditory brain stem response thresholds returned to preoperative levels by day 14 in

both techniques.

The endolymphatic sac communicates from the posterior fossa to the endolymph

space of the cochlea and vestibule. The inner ear can therefore be directly accessed via the

endolymphatic sac. This approach seeks to minimize direct trauma to the cochleovestibular

apparatus by remotely applying the biological substance. Successful delivery of adenoviral

vectors to the cochlea via the endolymphatic sac with minimal histological and physiological

disruption to the system has also been demonstrated (Yamasoba et al. 1999b). These data

have not been replicated in the cellular therapy literature.

As a treatment of spiral ganglion neuronal damage, the direct delivery of vectors or

cells to the auditory nerve is feasible. Access to the auditory nerve is routinely achieved in

the human patient for tumour removal and treatment of incapacitating vertigo (Brackmann

2004). The modiolus of the cochlea and the auditory nerve (Badi et al. 2002; Hillman et al.

2003) have been successfully accessed in animal models . NSCs injected in the basal

modiolus via cochleostomy, survived for two weeks and migrated as far as the apical

modiolus in cisplatin injured mice, but no retrograde migration of cells towards the brain

stem was noted (Tamura et al. 2004).

27

The potential for surgical trauma, inflammation and hearing loss associated with these

infusion or microinjection techniques has led to the investigation of a less invasive delivery

method to the cochlea. The potential to deliver a variety of vectors across an intact round

window membrane by loading vectors onto a Gelfoam patch that was placed in the round

window niche has also been explored (Jero et al. 2001a). Adenovirus and liposome vectors,

but not the AAV vector, effectively infected inner ear tissues as evidenced by detection of

reporter genes. Thus, diffusion across the round window membrane has been shown to be an

effective, atraumatic, but vector-dependent method of delivery for gene transfer vectors. Due

to the relative size of cells compared to the gap junctions in the round window membrane,

this technique would not be feasible for cellular therapy of the inner ear.

Lastly, successful intravenous delivery of NSC to target intracranial as well as

extracranial tumours has been demonstrated in mouse model (Brown et al. 2003). NSC

introduced into the tail vein of the mouse migrated and survived in tumour tissue with

minimal accumulation in normal tissues. Such a concept has not been described in the inner

ear biological therapy literature.

1.7 Pre-clinical Applications of Gene Therapy

The pre-clinical applications for gene transfer in the inner ear have focused upon

three broad areas. The first application was to demonstrate the protective effects of various

neurotrophins and growth factors in certain ototoxic scenarios. Secondly, gene therapy

technology has been used to introduce antioxidant gene over-expression as a technique of

ameliorating aminoglycoside-induced oxidative stress. Finally, gene transfer technology has

28

been used to induce the differentiation of non-sensory organ of Corti cells toward hair cell

fates, a preliminary step toward hair cell regeneration.

Certain neurotrophins and growth factors, including BDNF, NT3 and GDNF have

been expressed within the cochlea as transgenic products. These agents have served to

protect sensory hair cells and the primary auditory neurons against atrophy and degeneration.

Staecker et al. (1998) used a herpes simplex virus-1 (HSV-1) vector to deliver BDNF to the

inner ear and assessed its protective effect against neomycin. The gene therapy group

demonstrated a 94.7% salvage rate for SGNs, in contrast to a 64.3% loss of SGNs in control

animals (without the BDNF transgene). Interestingly, while BDNF expression was

ubiquitous in inner ear tissues, this was not the case for the reporter gene, β-galactosidase.

This reporter gene was detected in only 50% of the cells thus identifying the cells specifically

transduced by the HSV-1 vector. This transduction rate was sufficient to affect cochlea-wide

BDNF distribution and ensure 95% SGN survival. The authors speculate that SGNs must

require only a small number of BDNF-producing cells to ensure the survival of the entire

ganglion (Staecker et al. 1998).

Both in vitro and in vivo models have been used to test the protective effect of AAV

mediated BDNF expression (Lalwani et al. 2002). A significant survival of SGN in cochlea

explants transduced with AAV-BDNF and challenged with aminoglycoside relative to

controls was observed. Although direct expression of transgenic BDNF could not be

recorded, the vector’s ability to salvage SGNs was tested against a gradient of known BDNF

concentrations applied directly to the cochlea explants. The vector system was able to

achieve the same protective effects as 0.1 ng/ml of BDNF. However, this protective effect is

sub-therapeutic, as the most efficient dose was determined to be 50 ng/ml, a concentration of

29

BDNF that results in almost total SGN protection. In the in vivo experiment, animals infused

with AAV-BDNF with an osmotic minipump displayed enhanced SGN survival. The

protection from AAV-BDNF therapy was region-specific; there was protection at the basal

turn of the cochlea, but not the middle or apical turn. The authors proposed that this regional

selectivity is a pharmacokinetic phenomenon.

Neurotrophin-3 (NT-3) mediated protection against cisplatin-induced ototoxicity has

been documented using an HSV-1 derived viral vector. The efficacy of the vector was

established in an in vitro study, where HSV-1-mediated transfer of NT-3 (demonstrated by

production of NT-3 mRNA proteins and by reporter gene expression) conferred increased

survival to cochlea explants after cisplatin exposure (Chen et al. 2001). These HSV – 1

effects were confirmed in an in vivo model, where HSV-1 mediated transfer of NT-3 to

SGNs suppressed cisplatin-induced apoptosis and necrosis. The authors suggest that these

findings may not only be useful to prevent cisplatin-related injury, but may also provide

preventative treatment for hearing degeneration due to normal ageing (Bowers et al. 2002).

The efficacy of an Ad vector carrying the GDNF gene (Ad.GDNF) to protect against

a variety of ototoxic insults has been established (Yagi et al. 1999). When administered prior

to aminoglycoside challenge, Ad.GDNF significantly protects cochlea and vestibular hair

cells from cell death. Pretreatment with Ad.GDNF also provides significant protection

against noise-induced trauma and transient cochlea ischemia (Hakuba et al. 2003; Yamasoba

et al. 1999a). Finally, Ad.GDNF enhances SGN survival when administered 4 to 7 days after

ototoxic deafening with aminoglycosides (Sha et al. 2001).

Antioxidants represent a potential therapeutic tool to counter the destructive effects of

reactive oxygen species that are considered to be triggered by aminoglycoside induced

30

ototoxicity (McFadden et al. 2003; Takumida et al. 1999). Recently, inner ear gene therapy

has been used to corroborate the protective effects of antioxidants against aminoglycoside-

mediated ototoxicity (Kawamoto et al. 2004). Catalase and superoxide dismutase (SOD2)

were introduced into the cochleae of guinea pigs, which were subsequently challenged with a

kanamycin and ethacrynic acid. The catalase and SOD2 expressing animals preserved their

auditory brainstem thresholds and had significantly less hair cell loss when compared to

control animals.

Inner ear gene therapy has also been used to verify the molecular “switch”

responsible for turning on the genetic program for supporting cell differentiation toward a

hair cell fate. Math 1, is a basic helix loop helix transcription factor that is a master

regulator gene in hair cell differentiation during cochlea development (Bermingham et al.

1999; Mondain et al. 1998; Zheng and Gao 2000). The rationale of attempting to

phenotypically convert supporting, non sensory cells to hair cells using Math 1 gene over

expression was used (Kawamoto et al. 2003). Following adenoviral vector introduction,

Math 1 transgene expression occurred mostly in supporting cells. Furthermore, 30 to 60

days following surgery, rudimentary hair cells (identified by electron microscopy and

immunostained with Myosin VIIa) were observed in ectopic locations near the organ of

Corti. These “new” immature hair cells appeared to act as target sites for axon fibres that

extended over a 50-micrometer range toward the newly formed ectopic hair cells.

Similar results have been demonstrated by Shou et al (Shou et al. 2003). These results

have been extended by introducing Math 1 into supporting cells of deafened guinea pigs

through adenovirus transfection (Izumikawa et al. 2005). Transfected animals showed

phenotypically normal inner hair cells and a recovery of increased auditory brain stem

31

response levels to normal levels. These findings demonstrate the potential of gene

therapy to coax a damaged mammalian ear toward regeneration via phenotypic

transdifferentiation of non-sensory cells in the organ of Corti. If stem cells have been

demonstrated in the mammalian inner ear and indeed cells can be coaxed to become

functioning hair cells with the use of gene therapy, a valid question is, what then is the

place of cellular therapy in biological therapy of the inner ear?

An answer to the role of cellular therapy may lie in treatment of subacute and

more chronic injury of the inner ear and in the clinical translation of current knowledge.

Work to date using gene therapy has exclusively been using models of protection from

ototoxic insult or alternatively acute injury where typically the ear is injured, several weeks

later a therapy is administered and a response to that therapy studied. Common clinical

scenarios of deafness present as chronic injuries typically following either ageing

(presbycusis) or noise induced hearing loss, in this scenario the injury causes damage to the

inner ear over years. In such a situation, not only has hair cell death occurred, but the organ

of Corti has lost most of the supporting cell and other cellular architecture (Schuknecht

1974). Furthermore, spiral ganglion neuronal death produces empty canals of Rosenthal, the

place of normal residence for spiral ganglion neurons in human life (Schuknecht 1974). In

such a situation, cell replacement therapy could conceptually be a better form of therapy,

bridging the physical gap between neurons and hair cells by introducing cells, rather than

attempting to regrow and reconnect existing structures over considerable distances. Further

support for exploring the idea of cellular therapy, is that none of the current published work

in gene therapy and stem cell identification for the inner ear has been clinically translated

32

successfully into the human model. Therefore, alternative strategies for sensorineural

deafness treatment should be explored.

1.8 Cellular Therapy of the Inner Ear

Cellular therapy is a technique for introducing progenitor (stem) or fully

differentiated cells into damaged tissue with the aim of either replacing or repairing

dysfunctional/ dying cells. The introduced cells should ideally migrate to and integrate

into the damaged tissue. Modest success has been demonstrated with cellular

transplantation into the central nervous system. Grafted cells tended to migrate toward

pathological areas in the brain and spinal cord, in some instances integrating in a

meaningful manner to ameliorate disease processes such as Parkinson’s disease,

Huntington’s disease and spinal cord injury in animal models (Barkats et al. 1998;

Bjorklund et al. 2003; Bjorklund and Lindvall 2000; Bjorklund et al. 2002). The success

of this technique in the central nervous system provides a rationale for its application in

sensorineural damage of the inner ear following insults such as ageing, noise trauma and

ototoxicity.

1.8.1 Graft Sources for Cellular Therapy in the Inner Ear

Published data on cellular therapy of the inner ear have used various cell types as

graft material, examining the feasibility of introducing cells at different stages of

differentiation into the inner ear. The source of potential graft material will be an early

critical determinant in the success or failure of cellular therapy as a potential treatment

33

modality. The ideal source would be autologous, replenishable and have minimal

morbidity when harvesting the tissue.

1.8.2.1 Stem Cells

A stem cell is defined as a cell that is capable of both self-renewal and

differentiation (Temple and Alvarez-Buylla 1999). A totipotent stem cell can give rise to

cells of a whole organism and the term only applies to the zygote or blastocyst stage of

cell division. A pluripotent stem cell can generate many different cell types with limits

and usually refers to embryonic stem cells (Temple and Alvarez-Buylla 1999). The term

“multipotent” refers to a stem cell that can generate different cell types of the same tissue

from which the stem cell was derived; a cell at this stage is often referred to as a

progenitor cell as well (Gage 1998; Gage 2000). The terms and definitions used in stem

cell literature vary considerably and a consensus on appropriate use of terms has not yet

been achieved in the scientific community.

1.8.2.2 Neural Stem Cells (NSC)

Two separate groups provided the first evidence of the presence of NSCs in the

adult brain (Reynolds and Weiss 1992; Richards et al. 1992). Reynolds and Weiss

isolated cells from the striatum of an adult rodent brain. They demonstrated that these

cells could proliferate, forming large spherical clusters of undifferentiated cells which

were called neurospheres (Reynolds et al. 1992; Reynolds and Weiss 1992). These

neurospheres consist of proliferating cells as demonstrated by the incorporation of 3H-

34

thymidine. Presently there are three key requirements for a cell to be considered a neural

stem cell. The first requirement is the expression of nestin, an intermediate cytoskeletal

protein. Secondly, a cell must have multipotency, which in the setting of the nervous

system implies the capacity to give rise to cells with both neuronal and glial cell fates.

Finally the cell should have the ability to self-renew (Bjornson et al. 1999; Gage et al.

1995; Johansson et al. 1999; Morshead et al. 1994; Reynolds et al. 1992; Reynolds and

Weiss 1992; Richards et al. 1992).

Within the CNS, the subgranular zone of the dentate gyrus and the cells of the

subventricular zone of the caudate nucleus represent the two most extensively

characterized regions in which the presence of NSCs has been validated (Alvarez-Buylla

and Temple 1998; Gage 2000; Gage 2002; Temple and Alvarez-Buylla 1999).

The c17.2 cell line represents an example of a neural stem cell which is a

multipotent neural cell line generated via retrovirus-mediated v-myc transfer into murine

cerebellar progenitor cells (Snyder et al. 1992). The c17.2 cells contain a lacZ reporter

gene enabling easy identification. C17.2 cells successfully grow and differentiate into

neuronal and glial tissue throughout the central nervous system from newborn to adult

(Snyder et al. 1992).

1.8.3 Successful Cellular Grafts in the Inner Ear

Cells grafted into the inner ear to date, include embryonic stem cells, neural stem

cells (NSC), developing dorsal root ganglion, stromal bone marrow cells and adult

utricular maculae (Table 3 – see page 35). The rationale for the use of each of these

35

Table 3: Summary of Cellular Therapy of the Inner Ear

Animal Model Cell Source Cell Injection Cell Destination Reference

Rat Adult Hippocampal Tissue

Cochlear Wall, Round window, Oval window ST, SV, Organ of Corti Ito et al 2001

Avian Adult mouse utricular maculae chick otic vesicle sensory epithelium otic

vesicle Li et al 2003

Avian ES cell lines chick otic vesicles sensory epithelium otic vesicle Li et al 2003

Mouse Dorsal Telencephalon e11.5 PSCC, Cochlear wall

Perilymph & Endolymph spaces of vestibule, labyrinth,

cochlea, spiral limbus, SGN

Tateya et al 2003

Rat DRG e13/14 Cochlear wall ST, Modiolus, SGN Hu et al 2004

Chinchilla Autologous Bone Marrow

Round Window and modiolus

ST, SV, Lateral wall cochlea, Modiolus and

SGN Naito et al 2004

Mouse Dorsal Telencephalon e11.5 Round window ST, Modiolus Tamura et al 2004

Mouse ES cell line PSCC Vestibule and few cells in SM Tatsunori et al 2004

Mouse ES Cells PSCC Vestibule and few cells in SM Sakamato et al 2004

Guinea Pig Partially Differentiated ES Cells Round Window SM Hildebrand et al 2005

Gerbils Gerbil Embryo Hippocampus Round Window SM, Organ Corti Hakuba et al 2005

ES, Embryonic Stem Cell; PSCC, Posterior Semicircular Canal; SM, Scala Media; ST, Scala Tympani; SV, Scala Vestibuli

36

groups lies in the embryological relationship of the cell types to the inner ear. The first

three cell groups have a direct phylogenetic root to the sensory epithelia of the inner ear,

however, they rely on post mortem harvesting of cells and typically are low yield sources

of tissue requiring large numbers of animals to produce sufficient cells for

transplantation. Bone marrow cells, though not neuroectodermally derived and therefore

not directly related to the inner ear, have been demonstrated to undergo

transdifferentiation and form neural cells (Jiang et al. 2002). This source of cells

therefore, may be a potential source of graft material that can reliably address the issue of

supply that the other cell sources, at present, do not address. The ability to isolate a self-

renewing group of cells in the adult mammalian utricle was demonstrated by Li et al

(2003). The group demonstrated that these cells could be integrated into a developing

animal’s cochlea, thus validating the potential use of these stem cells as a graft material

for cellular replacement (Li et al. 2003a).

Embryonic stem (ES) cells are totipotent cells derived from the early embryo. ES

cells can be induced to differentiate toward a neuronal lineage and as expected appear to

be the most plastic cells available for experimentation. All three neural lineages can be

formed with both mammalian and human embryonic stem cells. Sourcing ES cells

requires transplanting cells into an enucleated oocyte, followed by developing the cells to

the blastocyst stage. Autologous transplantation is then possible by selecting, expanding

and differentiating the required cell population (Svendsen and Smith 1999). The

development of a method of reliably converting rapidly expandable ES cells into hair

cells could provide a potentially vast source of graftable cells.

37

Murine embryonic stem cells have been coaxed in vitro to develop into hair like

cells that express a comprehensive array of inner ear sensory epithelia marker genes and

phenotypic appearance (Li et al. 2003b). Furthermore, when genetically labelled ES cell

derived inner ear progenitors were introduced into the developing chick inner ear, the ES

cells that migrated toward and resided amongst developing sensory epithelia expressed a

similar array of key transcription factors and characteristic hair cell markers. Recent

experiments have demonstrated the feasibility of xenografting ES cells into the scala

media of the cochlea, vestibular sensory epithelia and auditory nerve of an in vivo

mammalian model (Hildebrand et al. 2005; Sakamoto et al. 2004).

Developing dorsal root ganglion (DRG) is a source of peripheral neurons that

have the ability to grow axons both peripherally and toward the spinal cord. In other

neuronal pathology models, transplanted DRG cells have developed functioning synaptic

connections. Histologically, sensorineural hearing loss is characterized by hair cell loss

and atrophy of the spiral ganglion neurons synapsing with the hair cell. DRG cells could

serve as replacement cells for damaged or dead spiral ganglion neurons, potentially

improving cochlea implant function which relies on functioning spiral ganglion neurons

to work effectively (Nadol and Xu 1992; Nadol et al. 1989). DRGs were harvested from

embryonic mice and successfully introduced into the adult rat cochlea (Hu et al. 2004;

Olivius et al. 2004). Significant cell survival was noted both in the scala tympani, with

cells congregating near the organ of Corti and amongst the spiral ganglion neurons.

Grafted cells not only survive in the cochleovestibular fluid system and spiral ganglion

for up to 10 weeks, but there was some evidence of axonal growth from implanted cells

toward the organ of Corti epithelia. These data are most significant in validating the

38

ability of cells introduced into the fluid filled spaces of the perilymph to migrate through

the osseous wall of the cochlea modiolus into the spiral ganglion; a feat previously

postulated from electron microscopy studies of the inner ear (Shepherd and Colreavy

2004).

The rationale of using Bone Marrow Stromal (BMS) Cells for transplantation lies

in the difficulty of obtaining large amounts of graft material when using neural sources

and the demonstration that BMS cells can undergo transdifferentiation into neurons

(Jiang et al. 2002; Jin et al. 2002). Furthermore, BMS cells could be readily obtained in

abundant amounts to be used as an autologous graft, eliminating rejection risk. Using

this rationale, autologous BMS were demonstrated to survive in the inner ear for up to 4

weeks (Naito et al. 2004). Cells were introduced into the scala tympani and modiolus

(containing the spiral ganglion neurons) via microinjection. Outcomes were similar to

NSC introduction, with cells mainly filling the perilymphatic space and spiral ganglion.

Immunohistochemically, about 1.2% of cells in the cochlea fluid chambers were GFAP

positive suggesting a glial phenotype and 0.8% of the cells surviving amongst the spiral

ganglion neurons expressed the neuronal epitope NF200.

Stem cells have been recently demonstrated in the adult mammalian utricular

maculae (Li et al. 2003a). These represent important data with relationship to both

hearing regeneration understanding as well as cellular therapy for the inner ear. Cells

from adult utricular epithelium were harvested and stained for Nestin (a stem cell

marker). Clonality of the cell was then shown as well as the ability to self renew, filling

all three criteria for stem cell definition. To demonstrate the ability of the cell to

differentiate toward hair cell fates, the stem cells were introduced into embryonic chick

39

otic vesicles. Labelled cells were identified amongst the sensory epithelia of the embryo

and these cells immunohistochemically stained for Myosin VIIa, illustrating that they

may be capable of forming hair cells.

1.8.4 Neural Stem Cells for Cellular Therapy of the Inner Ear

Neural stem cells are undifferentiated neural tube derivatives, hence their

potential plasticity and ability to integrate and differentiate into inner ear cells should

intuitively be greater than DRG cells, which are relatively more differentiated. It has

been postulated that NSC provide a more suitable cell graft material than other

alternatives for nervous system replacement therapy. Theoretically, NSC could better

incorporate into damaged nervous system; the replacement cells may reform neuronal

circuits more readily or recreate feedback loops. Furthermore, there is evidence that NSC

have an affinity for injured neurological tissue and may have intrinsic trophic factors

which allow them to survive more robustly in neural tissue (Bjorklund and Lindvall

2000; Brown et al. 2003; Kim et al. 2004; Ourednik et al. 1999; Ourednik et al. 2000).

In early work, neural stem cells from an embryonic rat brain were grown in a

typical neurosphere formation and immunophenotypically demonstrated Brn3c and

Myosin VIIa expression when introduced into the inner ear (Kojima et al. 2004). These

markers have also been identified in mature hair cells and thus could be considered as

preliminary evidence regarding the potential of central nervous system derived NSCs to

develop into mature hair cells.

NSCs introduced into damaged inner ears survive amongst most cellular

populations of the inner ear. The vast majority of cells transplanted into the scala

40

tympani of the cochlea remained in the perilymphatic space, lining the walls of the scala

tympani and vestibuli. NSCs however, do show a remarkable ability to migrate through

the fluid and tissue spaces of the ear. From the basal turn of the cochlea cells migrated to

the labyrinth epithelium, scala media, spiral limbus and modiolus. The grafted cells,

when they reached their final destination in the ear, occasionally took up some

morphological features of the host tissue including hair cell like phenotypes (Tamura et

al. 2004; Tateya et al. 2003). Immunophenotypically most cells stained positively for

GFAP (glial) and MAP2 (neuronal). Myosin VIIa (a hair cell marker) was only

identified in 5% of grafted cells located in the vestibule. No Myosin VIIa positive

transplanted cells were found in either the cochlea chambers or sensory epithelia.

1.8.5 Neural Stem Cells as Gene Therapy Vectors

In addition to their use for cellular replacement, transplantation of cells into the

inner ear may also be used as a delivery vehicle for proteins of interest, such as

neurotrophins or other growth factors. Cells engineered to produce genes and

subsequently neurotrophin protein products have been successfully introduced into the

ear (Iguchi et al. 2003). In grafted ears, elevated levels of neurotrophins were

demonstrated, illustrating the potential use of stem cells as long-term molecular factories,

with theoretical output capacities much greater than traditional viral or non-viral

methods.

41

1.9 Risks and Limitations of Intra-cochlea Biological Therapy

Major risk factors associated with the introduction of either genes or cells into the inner

ear are twofold: damage to the cochlea structure and function as a consequence of

delivery modality and the relative safety of the biological material transferred. Delivery

modalities that prevent damage to the cochlea structure/function have been described in

chapter 1.6. The safety of the gene transfer agent or cell is determined by assessing its

immunogenicity, toxicity and unwanted dissemination of the therapeutic agent outside of

the target region.

Utilizing AAV as the gene therapy vector, transgene expression within the

contralateral cochlea of the AAV perfused animal has been observed, albeit much weaker

than within the directly perfused cochlea (Lalwani et al. 1996). Subsequently, transgene

expression in the contralateral cochlea using Adenovirus has also been shown (Stover et

al. 2000). Expression of the transgene away from the intended target site, that is, within

the contralateral cochlea, raises concern about the risks associated with dissemination of

the virus from the target tissue. The appearance of the virus, distant from the site of

infection may be due to its hematogenous dissemination to near and distant tissues.

However, this is unlikely due to the absence of the viral vector in near and distant tissues.

Other possible explanations include migration of AAV via the bone marrow space of the

temporal bone or via the cerebrospinal fluid (CSF) space to the contralateral ear (Kho et

al. 2000). The perilymphatic space into which the virus is perfused is directly connected

to the CSF via the cochlea aqueduct; transgene expression within the contralateral

cochlea aqueduct has been demonstrated following introduction of the viral vector in the

42

ipsilateral cochlea (Stover et al. 2000). Collectively, these results suggest potential routes

for AAV dissemination from the infused cochlea via the cochlea aqueduct or by

extension through the temporal bone marrow spaces. Dissemination of grafted cells into

contralateral cochleae has not been addressed in the literature to date.

Although transgene expression within the inner ear has been well established,

several limitations of the gene transfer vector are evident. These include cell-target

specificity of the gene transfer agent and the sustained/regulated expression of the

transgene by the transduced cell.

Host vs. graft rejection of either introduced vectors or cells is a concern with any

form of homograft or xenograft. The ear is relatively immunoprotected by the blood

labyrinthine barrier (Lin and Trune 1997; Trune et al. 1997). Some histopathological

evidence is available in the literature to argue against hyperacute rejection of mouse ES

in the guinea pig cochlea. Following ES introduction, some fibrous tissue around still

viable ES cells 4 weeks following implantation was observed (Hildebrand et al. 2005).

The authors argued that this did not represent a significant reaction, as the ES cells still

remained fluorescent (i.e. viable). Lastly and probably of greatest concern is the

possibility of tumour formation with the introduction of dividing cells into the inner ear.

Significant overgrowth of cells or tumour formation following progenitor/stem cell

therapy of the inner ear has not been reported to date.

43

1.10 Neurobiology of the Olfactory Epithelium

1.10.1 The Olfactory Epithelium

The olfactory epithelium is a unique junction between the peripheral and central

nervous system, that continuously regenerates a single type of neuron throughout adult

mammalian life (Calof et al. 1996a; Calof et al. 1996b). Olfactory mucosa consists of

pseudostratified epithelium and lamina propria separated by a basement membrane. The

lamina propria consists of Bowman’s glands, ensheathing cells and axonal bundles. The

pseudostratified structure is composed of three distinct cell types: olfactory neuronal

cells, non – neuronal cells and basal cells.

Olfactory neurons are true bipolar neurons with cell bodies contributing to the

majority of the middle (intermediate) compartment of the olfactory epithelium. A

dendrite from the neuron terminates in cilia protruding into the nasal cavity and a single

axon from each neuron travels through the basement membrane being enveloped by

olfactory ensheathing cells then through the cribriform plate of the anterior cranial fossa

to make its first synapse in the olfactory bulb (Beites et al. 2005). Immature olfactory

neurons are bipolar and also contributed to the middle compartment of the epithelium,

however they do not have cilia or synaptic contacts (Calof et al. 1996a; Calof et al.

1996b).

The non-neuronal olfactory population consists of sustentacular (supporting) cells

and Bowman’s glands. Sustentacular cells are considered the glial or supporting cell for

olfactory neurons. They have a globose morphology with the cell body located apically

in the olfactory epithelium and a long stalk extending basally terminating in a contact

44

with the basement membrane (Andres 1975). Bowman’s glands are located in the

lamina propria of the olfactory epithelium. The glands secrete fluid that travels through a

duct and is expelled onto the surface of the mucosa (Andres 1975; Tos and Poulsen

1975).

Basal cells lie adjacent to the basement membrane and are divided into two

groups: horizontal and globose basal cells. Horizontal basal cells were once thought to

be the progenitor cells for olfactory neurons and the globose basal cells were thought to

be intermediate neuronal precursors (Calof and Chikaraishi 1989; Mackay-Sim and Kittel

1991; Yamagishi et al. 1989). Later work provided evidence for the globose basal cell

being the precursor to the olfactory neuron (Caggiano et al. 1994; Schwob et al. 1994).

Current work on identifying the progenitor for the mature olfactory neuron fails to reach

a consensus on which of the two morphologically different cells is the precursor.

Furthermore, there is some evidence that surrounding environmental niche structures are

just as critically involved in the process of olfactory neuronal development (Beites et al.

2005; Calof et al. 2002; Gordon et al. 1995; Kawauchi et al. 2004; Murray and Calof

1999). Recently therefore, in an attempt to better define and identify the olfactory

progenitor cell, research efforts have been directed towards identifying unique molecular

markers of the olfactory progenitor cell.

1.10.2 Olfactory Progenitor Cells

Mature olfactory neurons of adult rodents are the result of mitosis that occurs in

the basal compartment of the olfactory epithelium. Unidentified stem cells produce

daughter cells which are identified both in vitro and in vivo by the expression of a

45

proneural gene Mash 1 (Beites et al. 2005; Manglapus et al. 2004). The daughter cells of

the Mash 1 progenitors express neurogenin 1, another proneural gene (Calof et al. 2002;

Gordon et al. 1995). The progeny of the neurogenin 1 cells, often called “intermediate”

cells undergo maturation in the form of terminal differentiation to form mature olfactory

neurons, which are identified by several specific neuronal markers such as neural cell

adhesion molecule and neural specific tubulin (Beites et al. 2005). Using Mash 1 -/-

knockouts, progress is being made into better identifying the putative stem cell that

resides in the basal region of the olfactory epithelium.

Mash 1 -/- knockout mice fail to develop intermediate and mature olfactory

neurons due to genetic interruption of the olfactory maturation pathway (Murray et al.

2003). These mice do, however, have a full complement of neural progenitor cells in the

basal layer of olfactory mucosa, which produce expanded expression of certain critical

neural primordial genes. Sox 2 is a homeobox transcription factor which is expressed in

many varieties of primordial neural stem cells, suggesting it may be a marker of a stem

cell pool (Beites et al. 2005; Kawauchi et al. 2004). In Mash 1 -/- knockout mice, Sox 2

is expressed widely throughout the expanded pool of basal cells.

Animals in which retinoic acid production is depleted show craniofacial

malformations as well as olfactory epithelium absence (Dickman et al. 1997; Schneider et

al. 2001). Tissue specific expression of retinaldehyde dehydrogenase (RALDH) appears

to be critical in determining where and when retinoic acid functions (McCaffery and

Drager 2000). Using in vitro cultures of olfactory progenitor cells and identifying

RALDH substrates using fluorescence, retinoic acid signalling via the RALDH 3

receptors appears critical for olfactory epithelium development (Beites et al. 2005).

46

Taken collectively these data suggest that the putative olfactory stem cell may express

high levels of Sox 2 and RALDH 3.

1.10.3 Olfactory Progenitor Cells Grown in Vitro

Under well controlled conditions, dissociated neurogenic regions of the CNS have

been isolated as single cells and grown in culture. These single cells then grow into cell balls

of undifferentiated progeny referred to as neurospheres (Gritti et al. 1996; Reynolds et al.

1992; Reynolds and Weiss 1992). The defining characteristics of these in vitro NSC cultures

are that they are clonally derived from a single cell, can differentiate into different neural cell

types and upon dissociation of primary neurospheres, new clusters of secondary neurospheres

are formed (Gritti et al. 1996; Reynolds et al. 1992; Reynolds and Weiss 1992). The

characteristics of neurospheres have been validated for single cell dissociated cultures of the

basal layer of rat olfactory epithelium, the author proving evidence that these newly

developed cultures contain olfactory progenitor cells (OPC) and proposing that these

olfactory “ball” cultures be referred to as olfactory neurospheres (Khan 2003). Whilst

neurospheres exhibit similar characteristics to neural stem cells, an important caveat to

consider is that the two may represent different cell types along a similar lineage pathway

and therefore neurosphere cells should not at this stage be referred to as neural stem cells.

1.10.4 Nestin in Olfactory Epithelium and Olfactory Spheres

As discussed in section 1.8.2.2., Nestin is an intermediate cytoskeletal protein, which is

currently the classic marker of neural stem cells. Nestin has been demonstrated in

pancreatic tissue (Hunziker and Stein 2000; Zulewski et al. 2001). Furthermore, some

47

neural progenitor cultures fail to express Nestin (Steindler et al. 1996). Within adult

rodent olfactory epithelium, nestin is expressed in sustentacular cell bodies and end feet

as well as in Bowman’s gland epithelial cells of the basal layer, but not in globose basal

cells, which are still considered the most likely olfactory stem cell candidate (Doyle et al.

2001). Taking these data collectively, therefore, although nestin immunoreactivity forms

the current basis of identifying neural progenitor and stem cells and is the marker selected

to identify olfactory progenitor cells in this thesis, it is by no means a definitive marker of

neural stem cell identity in the olfactory epithelium. Certainly, the existence of stem cells

not labelled by nestin, perhaps more proximal in the lineage pathway, cannot as yet be

excluded.

1.11 Aims of This Thesis

To advance research in biological therapy for the inner ear there is a need for the

development of both in vitro and in vivo systems to test potential new therapies. Testing

cellular therapies does not translate well when using currently established in vitro models

that have been historically used to test gene therapy. Thus, the first aim of this thesis is to

develop an in vitro model for cellular therapy that provides some advantages over the

current flat inner ear explant model.

Furthermore, to clinically translate cellular therapy of the inner ear to the human

model, a graft cell that is potentially homologous and easily harvestable with low

morbidity would be of considerable advantage over the currently explored post mortem

homo/ xenograft derived graft cells. The olfactory epithelium contains stem/ progenitor

48

cells that could potentially serve as such an ideal graft cell. The second part of this thesis

aims to examine the ability of mouse ONS/ OPCs to survive and populate the inner ear,

as a future potential therapeutic graft cell. Mouse olfactory epithelium will be harvested,

purified and tested for their ability to form olfactory neurospheres. Furthermore, ONS/

OPCs will be tested in the new in vitro model and the work extended to a mouse in vivo

system where OPCs will be compared to a well established stem cell line c17.2, in their

ability to survive following implantation. The goal of this thesis, therefore, is to advance

our understanding of the potential of cellular therapy in treating sensorineural hearing

loss.

49

Chapter 2 Materials and Methods

2.1 Animal Surgical Methods

2.1.1 Harvesting and Preparation of the Cochleovestibular Explant

All animal care was in accordance with the committee on Animal Care Guidelines for

Humane Treatment of laboratory animals (New York University Approval Number 030707-

01). Postnatal day 1 to 2 CD1 mouse pups were used for explant harvesting (parent animals

from Charles River Laboratories Inc., Wilmington, MA.). Mice were sacrificed by

hypothermic anaesthesia and decapitation.

Under microscopic vision, the skin and soft tissue of the head was removed. Next,

the superior half of the cranium was sharply removed and brain eviscerated. The trigeminal

nerve was sharply dissected off the floor of the middle cranial fossa to reveal the

cochleovestibular complex and stump of the basal component of the spiral ganglion complex

as it entered the cochlea modiolar base. The whole cartilaginous cochleovestibular complex

was then removed in toto by sharp dissection from the middle cranial fossa floor dissecting in

an extracapsular fashion. Excess basal spiral ganglion was then transected perpendicular to

the nerve as it entered the modiolus of the cochlea. Two thirds of the superior semicircular

canal was resected to gain clear access to the common crus and allow egress of excess cells

and intra-cochlea fluid during vestibular injection. Finally, one third of the cartilaginous otic

capsule was removed and a wedge of cochlea basal turn was resected by sharp dissection to

allow microinjection access to the scala tympani, simulating the in vivo scenario.

50

Twelve explants were cultured in c17.2 cell culture media to observe their survival.

Two explants were fixed every 2 days from day 0.25 to day 8.25 to observe architectural

integrity (see section 2.3).

2.1.2 Stem Cell Introduction into In vitro Models

To test the ability of cells to migrate from the surface of the explant to within the

cochleovestibular structure, a direct application technique was employed. 1 micro liter of

NSCs in a concentration of 1 million cells/ mL, giving an approximate total cell count of 100

cells, was microinjected directly over the open vestibule, basal cochlea and basal spiral

ganglion structures of cochleovestibular explants. The explants were then returned to the

incubator, left undisturbed for 2 days and the explants removed, fixed and processed.

To evaluate stem cell survival after direct microinjection into the explant, stem cells

were microinjected into the superior semicircular canal and a cochleostomy site in the basal

turn of each explant (Figure 1 – see page 51). Borosilicate glass capillary tubing (Warner

Instrument Corp.) was fashioned, with a bunsen burner, to form straight and curved

microinjection pipettes with < 0.5 mm outer diameters. Oil/ water calibration was used to

quantify microinjection amounts using the microinjection manipulator (Sutter Instruments

Corp.). Calibration allowed specific volumes (and therefore measured numbers of cells) to

be introduced into the 3 injection sites of the cochlea and vestibule; 600 nanolitres into the

vestibule, 300 nanolitres into the perilymphatic space of basal cochlea and 200 nanolitres

onto the basal spiral ganglion. Each injection was performed by microinjection over one

51

Figures 1A, 1B and 1C

52

to two minutes, depending on the volume introduced. Following microinjection, explants

were returned to a 37 °C (5% CO2) incubator for up to 8 days culturing.

To determine whether cell growth phase affects cell number survival in cochlea

explants, c17.2 cells were introduced at various stages of confluence. Cells grown to 50%,

70% and 100% confluence were harvested by trypsinization (see section 2.2.1). Each

suspended cell solution was spun down, supernatant was aspirated and the cell pellet

resuspended in PBS solution to a concentration of 1 x 10 6 cells/ mL. Fixed volumes of cells

as detailed above were injected into eighteen explants (six per stage of confluence) and the

cochleae left at 37 °C (5% CO2) for 6 days. Media were changed every second day. After 4

days, the media were removed and the explants washed twice for 2 minutes in PBS solution.

PBS was then aspirated and the explants fixed and processed as described below.

To determine optimal cell numbers for introduction, various cell concentrations were

assayed. Three concentrations were chosen – 1 x 105 cells/mL, 1 x 106 cells/mL and 2 x 106

cells/mL. A negative control where no cells were introduced was included. Cell culture and

preparation was undertaken as described in section 2.2.1. Each cell concentration was

injected into six explants (three different sites) and assayed for 4 days. Media were changed

every second day. After 4 days, the media were removed and the explants washed twice for

2 minutes in PBS solution. PBS was then aspirated and the explants fixed and processed as

described in sections 2.3, 2.4 and 2.5.

To clarify the maximum duration of cell survival in explants, a time assay was

performed. Six explants were injected with c17.2 cells grown to 70% confluence and at a

concentration of 1 x 106 cells/mL for each time point. Five time points were chosen – 6

hours (0.25 days), 2.25, 4.25, 6.25 and 8.25 days. A negative control was included where no

53

cells were introduced. Media were changed every second day. At the end of each time point

the media were removed, explants were washed twice in PBS, fixed and processed as

detailed in sections 2.3, 2.4 and 2.5.

To assay the effect of ototoxins upon the explants, the cochleovestibular structures

were incubated with varying concentrations of ototoxins. Ototoxin concentrations that

reliably damaged cochlear and vestibular explants have been previously reported (Clerici et

al. 1996; Forge and Li 2000; Zheng and Gao 1999). The ototoxin concentrations used in this

study included Gentamicin 0.1, 1 and 10 mM; as well as Cisplatin 50 mM and 0.2 M.

Negative controls where no ototoxin was applied, were included. Explants were maintained

for 48 hours and then washed twice in PBS, fixed and processed as detailed in sections 2.3,

2.4 and 2.5.

To assay the effect, if any, of neural stem cell survival in explants following ototoxic

injury, explants were pre-treated with various concentrations of Gentamicin and Cisplatin for

24 hours, prior to c17.2 NSC transfer. Six explants were treated with each concentration of

Gentamicin at 0.1 mM and 1 mM as well as Cisplatin at 50 mM. Negative controls where no

ototoxin was applied but cells were introduced, were also used.

Before c17.2 NSC introduction, the explants were washed twice with PBS for 5

minutes, to remove any residual ototoxin. Cells were trypsinized at 70% confluence and

counted to a concentration of 1-million cells/ mL and then introduced into the explants at the

volumes previously stated. Media were changed every second day. At the end of four days

the media were removed, explants were washed twice in PBS, fixed and processed as

described in sections 2.3, 2.4 and 2.5.

54

To document the effect, if any, of growth factors on the survival of c17.2 NSCs in

explants, various growth factors were applied to the culture. Explants were either co-treated

with NT3 5 ng/ mL (Invitrogen Corp.), BDNF 0.1 ng/mL (Invitrogen Corp.) or NT3 plus

BDNF when the c17.2 cells were introduced into the explant model. Six explants for each

combination of neurotrophins were injected with cells. A negative control was included that

comprised of untreated explants injected with c17.2 cells. A six-day assay was performed

with a 1 x 106 cells/mL concentration of c17.2 cells introduced into the explants at the

volumes previously stated. Media were changed every second day, with the various

combinations and concentrations of neurotrophins included in the media. At the end of 6

days, the media were removed and the explants were washed twice in PBS, fixed and

processed as described in sections 2.3, 2.4 and 2.5.

To determine the survival of ONS/ OPCs in explants, specific volumes and therefore

cell numbers were introduced into the three same injection sites of the cochlea and vestibule.

Following microinjection, explants were returned to a 37 °C (5% CO2) incubator. An assay

was performed using either preferential OPC (Neurobasal media, Invitrogen Corp., Carlsbad,

CA, USA) or explant media (Advanced MEM, Invitrogen Corp., Carlsbad, CA, USA)

changed every two days. The explants were maintained for up to 8 days in culture. Explants

were then fixed and processed as described in sections 2.3, 2.4 and 2.5.

2.1.3 Olfactory Epithelium Harvesting

Olfactory turbinates were isolated from postnatal (P0 to P2) ubiquitously expressing

GFP mice (a gift from Dr Okabe) (Okabe et al. 1997). To establish that the mated population

55

was in fact ubiquitously expressing GFP, all pups were phenotypically checked under

ultraviolet light. The presence of a glowing coat and fluorescing tail confirmed the GFP

population for olfactory epithelium harvesting. To establish that the nasal mucosa was

expressing GFP, a sample pup was sacrificed and the sinonasal complex dissected in toto

from the remainder of the maxillofacial complex. This sample was subsequently sectioned

and processed as described in sections 2.3 and 2.4, to view under immunofluorescence.

With the identified GFP pup population, animals were killed by hypothermic

anaesthesia and decapitation. The olfactory turbinates were sharply dissected under

microscopic vision from the lateral wall of the nose, ensuring that no sinus bone, septal

cartilage and infratemporal fossa musculature were incorporated into the dissected specimen

of respiratory and olfactory mucosa. To maximise the harvest of olfactory epithelium, all the

mucosa from the superior two thirds of the septum was also microscopically dissected. All

the harvest tissue was then placed in ice cold Neurobasal Solution (GIBCO Corp.) with 9.6

mg/ml HEPES. The dissected turbinates were then centrifuged at 700 x g for 10 min. The

supernatant was decanted and the turbinates minced with fine scissors. The subsequent

olfactory cell culture protocol is described in sections 2.2.2.

2.1.4 GFP OPC Introduction into the In Vivo Adult Mouse Model

CD1 adult male mice (35 – 40g) were anaesthetised by intraperitoneal injection of

Ketamine (100 mg/kg) and Xylazine (9mg/kg) and placed on the operating table supine,

core body temperatures were maintained with a warming operating table cover. The right

tympanic membrane was initially micro-inspected to exclude infection in the middle ear

56

space. If the ear was disease free, a post auricular approach to the cochlea and round

window was performed under microscopic vision. The right bulla was opened with

microdrill and the round window perforated with a fine otologic micropick. Entry into

the inner ear was confirmed by perilymph being visible in the round window niche. An

angled glass micropipette needle (outer diameter < 0.5 mm) was inserted into the round

window, no attempt was made to aspirate fluid and 1000 nL of cell suspension

(approximately 1000 cells) was microinjected over 2 minutes into the cochlea. The

volume used overfilled the explants and some spillage deliberately occurred. Previously

divided digastric muscle was then harvested and placed into the round window niche and

the wound closed in layers. Six animals per study group were operated upon, including

c17.2, OPC and control groups. The negative control group was operated upon, by

delivery of PBS instead of GFP OPCs into the cochlea. During recovery period, animals

were given Buperonex (a Non steroidal anti-inflammatory drug) for analgesia in the first

24 hours post operatively.

2.2 Cell Culture Methods

2.2.1 NSC C17.2 – Growth and Preparation Assays

C17.2 cells (a gift from Dr E. Snyder, Harvard University, Boston, MA, USA) were

used for all in vitro experiments. C17.2 cells are a beta galactosidase expressing neonatal

mouse cerebellum derived neural stem cell line, described in section 1.8.2.. Cells were

57

thawed in the vial by gentle agitation in a 37°C water bath. Cells were then pipetted into a 10

cm dish containing 9.0 mLs of feeding medium and triturated gently. Cells were grown in

0.1% gelatin coated plates with Dulbecco’s Modified Essential Medium (GIBCO Corp.),

supplemented with 10% Foetal Bovine Serum (GIBCO Corp.), 5% Horse Serum (GIBCO

Corp.), GlutaMAX (2mM L-glutamine), Penicillin, Streptomycin and Amphotericin (25

nanograms/mL) (GIBCO Corp.). Cells were grown in 10 cm petri dishes (Corning Corp.)

and initially incubated at 37°C, 5% CO2-air incubator. After 4-8 hours in the incubator,

when the cells settled and attached, the medium was changed completely and replaced with

fresh medium. During incubation, the media required changing every second day and cells

required subculturing every second to third day.

For subculturing, cells were washed with PBS to remove traces of serum and then

0.65 ml of 0.05% Trypsin-EDTA solution was added to the dish, which was then incubated at

37°C for 2 minutes. Then cells were observed under an inverted microscope until the cell

layer was dispersed. Three to five millitres of feeding medium was then added to inactivate

the trypsin and the suspension was pipetted and triturated. Cells were then centrifuged for 3

minutes at a 1000-rpm, the supernant was removed and the cells resuspended in 10 mLs of

PBS. A subcultivation ratio of 1:10 produced a phenotypically stable and sustained

population of c17.2 cells.

On the day of surgery, cells of an appropriate confluence were washed twice with

Phosphate Buffered Saline (PBS) solution (GIBCO Corp.), trypsinized with 1 mL of 0.05%

Trypsin/ EDTA (GIBCO Corp.) and incubated for 2 minutes at 37 °C. Trypsinization was

stopped, cells were then triturated and spun down. In preparation for transplantation, cells

were resuspended in PBS to the appropriate concentration and 10 µL removed for cell

58

counting. The 10 µL suspension was stained with 0.5 µL of 0.01% Trypan Blue, left to react

for 1 minute and then cells counted to verify the required cell concentration using a standard

hematocytometer.

2.2.2 Olfactory Neurosphere Isolation and Culture

2.2.2.1 Respiratory and Olfactory Tissue Size Fractionation

This protocol was modified from a similar protocol used to harvest olfactory

progenitor cells from rat pups (Khan 2003). Dr Khan’s work was originally based on

protocols used to identify basal cells from olfactory epithelium (Cunningham et al. 1999;

Ronnett et al. 1991). The minced respiratory and olfactory epithelial tissue, described in

section 2.1.3, was enzymatically digested for 1 hr at 37ºC with agitation in a cocktail of

enzymes: 4 mg/ml dispase II, 1 mg/ml collagenase, 1 mg/ml hyaluronidase and 60 µg/ml

deoxyribonuclease I; all dissolved in HEPES–Neurobasal solution containing 1% (w/v)

BSA. The digested cell suspension was filtered through a wire mesh with openings of

229 µm (Small Parts Corp., FL.). The filtered cell suspension was centrifuged at 700-x g

for 10 min. The pellet was resuspended and triturated gently in Neurobasal medium

containing 10% FBS, 100 U Penicillin G, 50 µg/ml gentamicin sulphate, 2.5 µg/ml

Amphostat B, 20 mM L-Glutamine. The cell suspension was passed through a second

filtration step of 40 µm cell strainer followed by a 10 µm nylon mesh filter (Millipore

Corp.). The nylon mesh was placed in the previously mentioned Neurobasal medium,

however 10% FBS was replaced with B27 supplement with addition of 20 ng/ml EGF

and 20 ng/ml FGF-2. These media were designated growth media. The trapped cells

59

were dislodged by gentle trituration using a fire polished Pasteur pipette. The dislodged

cells were then centrifuged at 700 x g for 10 minutes. The supernatant was decanted and

the pellet went through a second wash by triturating with fresh growth medium. The

solution was spun again and the pellet was mechanically dissociated by trituration 40–50

times. Single cells were then plated onto multi-well chamber slides without laminin and

incubated at 37ºC in the presence of 5% CO2. Half of the growth medium was replaced

with fresh medium every 3 days. This method favoured the isolation of olfactory

neurons, olfactory ensheathing cells and other basal layer cells that may contain

presumptive olfactory progenitor cells.

2.2.2.2 Passaging and Nestin Staining of Olfactory Spheres

To confirm olfactory progenitor cell passaging, cells were grown on 2-well Lab-

Tek chamber slides without laminin and incubated at 37ºC in the presence of 5% CO2 for

6 days. Half of the growth medium was replaced with fresh medium after 3 days. The

olfactory culture was then subjected to trypsinisation at day 6, with trypsin pre-incubated

at 37ºC water-bath for a minimum of 2 hr. First, the cell culture medium was washed

with PBS and trypsin was added to the cells and incubated for 20 min at 37ºC. The

dislodged cells were centrifuged at 700-x g for 10 min. The pellet was resuspended in 5

ml normal growth medium and further dissociated mechanically with a fire-polished

Pasteur pipette. A serial dilution was performed by removing 1 ml of the dissociated

cells and adding 4 ml of progenitor growth medium. This was repeated twice more. The

diluted cells were plated in a 96-well plate. Individual wells were checked for the

presence of clumps of cells and eliminated from the experiment. Only wells with single

60

cells or no cells, were identified to be studied. The 96-well plate was incubated at 37ºC

in the presence of 5% CO2 and the cells were analysed using an inverted microscope

(Olympus Corp.) once a week. The medium was only changed once every two weeks

only in the first month of their growth period, by changing only 50% of the medium. The

cells were maintained in the incubator for one and a half months using this technique,

with sample cells plated weekly to check for Nestin positivity.

Fixation and immunohistochemical techniques were performed as described in

sections 2.3 and 2.4.

2.2.2.3 Differentiating Olfactory Spheres

The olfactory progenitors were grown as previously described (Section 2.2.2.1).

After 6 days the growth medium was replaced with differentiating medium consisted of

growth medium and 2% FBS, without addition of the growth factors EGF and FGF-2.

Half of the growth medium was replaced with differentiating medium, this process was

repeated 3 times to dilute the growth factors to a negligible concentration and at the same

time to wash the cells. The cells were allowed to differentiate for 48 hours at 37ºC with

5% CO2. Cell fixation and immunofluorescence were performed as described in sections

2.3 and 2.4. Primary antibodies used were monoclonal anti-β-tubulin and polyclonal

anti-GFAP. Fluorescence was detected using Alexa secondary antibodies.

2.2.2.4 Preparing Olfactory Neurospheres for Microinjection

ONSs were maintained for 6 days in preparation for cell transplantation. On the

day of transplantation, cells were trypsinized, inactivated with growth media and spun at

61

700-x g for 10 minutes. Supernatant was discarded and the pellet resuspended in PBS to

provide cell counts of 1 x 105 cells/mL to 1x 106 cells/mL, which were considered

acceptable for in vitro and in vivo microinjection. Counts higher than this resulted in

occluded microinjection needles, as ascertained by the c17.2 microinjection work.

2.3 Fixation Methods

To determine the best fixation method for explants, they were fixed by glass fine bore

microinjection of 4% Paraformaldehyde (PFA) solution (adjusted to pH 7.4 with Sodium

Hydroxide) into the cochleostomy of each explant. A fixative assay was then performed with

explants maintained for 15 minutes, 30 minutes, 2 hours at room temperature and finally

overnight at 4 degrees Celsius. Subsequent explant fixation was performed according the

most appropriate outcome from the fixative assay.

To fix the OPCs prior to Nestin staining, 4% Paraformaldehyde (PFA) solution

(adjusted to pH 7.4 with Sodium Hydroxide) at room temperature was applied for 15 minutes

then the cells were washed twice with PBS and processed for immunohistochemistry as

described in sections 2.4 and 2.5.

Fixation of the in vivo model was carried out by cold intracardiac perfusion of 4%

Paraformaldehyde (PFA) solution (adjusted to pH 7.4 with Sodium Hydroxide). The animals

were anaesthetised by intraperitoneal injection of Ketamine (100 mg/kg) and Xylazine (9mg/kg)

and placed on the operating table supine. A bilateral thoracotomy was performed, followed by a

pericardiotomy to access the aortic root. A silk purse string was placed around the aorta and the

vessel cannulated with an 18-gauge needle. A decompression incision was made in the right

62

atrium and the animal infused firstly with cold PBS for two minutes then for two minutes with

cold 4% PFA. Fixation was confirmed by palpating for abdominal and spinal rigidity.

Following PFA perfusion, cochleae were harvested by sharp dissection and the stapes removed

from the oval window niche. Cold PFA was flushed through the cochleae via the oval window

and then the specimens decalcified in EDTA for two weeks. Following decalcification, the

cochlea was cryprotected in 30% sucrose for 1 hour at room temperature and then embedded in

OCT (Sukora, Japan) cryosection media, frozen to –50°C and cryosectioned 7 µm thick at 42 µm

intervals and mounted on slides. Explants were similarly cryoprotected, frozen and sectioned 5

µm thick at 30 µm intervals. Slides were then air dried for 1 hour and frozen at –80°C in

preparation for future immunohistochemistry or observation as described in sections 2.3 and 2.4.

2.4 Staining and Immunohistochemical Methods

Following fixation, explants were washed twice with PBS for 2 minutes and then

incubated for 2 hours at 37 °C in Xgal staining solution (Stratagene corporation). Following

Xgal staining, the specimens were rewashed twice with PBS solution and cryoprotected with

30% sucrose for 2 hours at room temperature. Specimens were then embedded in OCT

cryosection media, frozen at –50°C and then cryosectioned 5 µm thick at 30 µm intervals and

mounted on slides. Slides were then air dried for 1 hour and frozen at –80°C in preparation

for future immunohistochemistry.

Before immunohistochemistry, slides were rewashed twice for 2 minutes in PBS and

then immersed in Triton X-100 0.1% in PBS for 5 minutes to permeabilise the tissue.

Sections were then washed twice with PBS for two minutes and blocked with I-Block

63

commercial blocking solution (Tropix Inc. Bedford, MA) for 20 minutes at room

temperature. The I-block was then tipped off and the primary antibodies applied. Primary

antibodies included beta galactosidase primary antibody (J1E7 – Iowa hybridoma Bank) at a

dilution of 1:250, Nestin primary antibody (rat 401 – Iowa Hybridoma Bank) at a dilution of

1:100, monoclonal anti-β-tubulin antibody at a dilution of 1:100 (Abcam Inc., Cambridge,

MA USA) and polyclonal anti-GFAP at a dilution of 1:1000 (Abcam Inc. Cambridge, MA,

USA) was applied for 1 hour at room temperature. Slides were then rewashed twice with

PBS for 2 minutes and Texas Red or Fluorescein antimouse antibody (Vector Laboratories,

Burlinghame CA) was applied at a dilution of 1:500 for 45 minutes at room temperature.

The final slide was then rewashed twice with PBS for 2 minutes and the slide cover-slipped

using florescence enhancing oil mount medium (Vector Laboratories, Burlinghame CA).

The coverslipped slides were then stored at 4°C in a dark refrigerator prior to viewing.

2.5 Microscopy and Digital Imaging Methods

Florescent viewing was carried out using a direct fluorescence microscope, black and

white digital camera (Zeiss Corp.) that allowed bright field and florescence image capture

and post image capture colouring software. Post imaging resizing and reformatting was

undertaken with Adobe Photoshop 7.0 (Adobe Inc.) and a Macintosh G4 computer (Apple

Computer, Cupertino CA).

Expression of the β-gal marker gene served to distinguished the c17.2 cells from the

surrounding endogenous cells. These cells were identified either under light microscopy by

64

blue staining, or alternatively under immunofluorescence microscopy by the fluorescence of

the secondary antibody. OPC/ OPC derived cells were identified by either GFP fluorescence

(OPC derived) or GFP fluorescence in combination with Nestin antibody

immunofluorescence (OPC). Survival of the implanted cells with all experiments was

assessed quantitatively within the vestibule and the cochlea and the results illustrated through

tables and/or photomicrographs.

For each experimental in vitro and in vivo arm, six explants or animals per group

were evaluated. Six sections were taken at 5 µm (for explants) and 7 µm (for in vivo

specimens) from the mid modiolar and paramodiolar regions, spacing slices at 30 µm for the

explants and 42 µm for the in vivo specimens. The mean cell counts presented in tables 4 to

8 and table 10, therefore, represent a mean from 36 sections (6 sections from each of six

animals). Cell counts from each of five regions; vestibule, apical, mid and basal regions of

the cochlea and basal spiral ganglion were taken directly at 16 - 40 x magnification under

light/ immunofluorescence microscopy using a standard click hematocytometer (Reichert,

Buffalo, NY, USA) and lots of patience.

2.6 Statistical Methods

For determination of sample size for in vitro and in vivo experiments, it was estimated

that at least six animals/ explant specimens were required for each study group (for a Type I

error of 0.05 and a 90% probability of detecting a true difference). These estimates were

obtained from pilot studies of surgery performed on animals prior to experimentation and

using previous laboratory data on gene therapy surgery in mice (Jero et al. 2001b).

65

Assuming a 20% loss of data due to technical reasons (animal sickness or death, processing

difficulties, etc.), eight animals / explants were included for each time point in each study

group.

Cell counts for six samples from each study group were added together, averaged and

a standard deviation determined. The averaged data were tabulated for each assay group. All

statistical comparison was made using the non-parametric Kruskal-Wallis analysis of ranks

with post hoc Tukey test, the p value was set at < 0.05 to achieve significance.

66

Chapter 3 an In vitro Model For Cellular Therapy of The

Inner Ear

3.1 Introduction

Most published cellular therapy data for the inner ear to date has focused on survival

of grafted cells, their distribution pattern within the inner ear and immunophenotypical

characteristics post-implantation (Iguchi et al. 2004; Iguchi et al. 2003; Li et al. 2004; Lopez

et al. 2004; Naito et al. 2004; Nakagawa and Ito 2004; Sakamoto et al. 2004; Tamura et al.

2004; Tateya et al. 2003). There is significant variability in models and methods used to

examine the feasibility of inner ear cellular therapy. Different animal models, cellular graft

material, techniques of introduction and outcome measures have been employed. All of

these differing approaches highlight the considerable effort that is required to evaluate each

variable and its potential outcome. The use of a suitable in vitro model may expedite the

rapid characterization of variables affecting stem cell transfer to the inner ear. Not only

would a suitable in vitro model potentially help predict the best cell choice and optimal cell

numbers for transplantation, but such a technique could also be used to rapidly test potential

facilitators and modulators of the survival and implantation process such as neurotrophins.

The feasibility and techniques of in vitro inner ear cellular therapy experimentation is

relatively uncharted. The ability of beta galactosidase labelled NSCs to survive within

utricular epithelium has been demonstrated, however these cells failed to successfully survive

67

within the organ of Corti epithelium (Fujino et al. 2004). Using this flat explant model

traditionally used for ototoxic and gene therapy assessment highlights some of the flaws of

translating the old model for cellular therapy. In particular, this old in vitro model could not

be used to determine optimal cellular concentrations and furthermore could not allow

determination of patterns of cell survival and integration in the various compartments of the

inner ear.

A neural stem cell line c17.2 (described in section 1.8.2.2) will be used as the

experimental cell for this part of the thesis. This cell has been previously shown to have a

tropism toward damaged neurological tissue (Riess et al. 2002; Tang et al. 2003). In fact, the

c17.2 cell has been shown to have the ability to migrate when injected intravenously, across

the blood brain barrier to reach damaged neurological cells in both the ischaemic and tumour

models (Brown et al. 2003; Kim et al. 2004). The c17.2 cell therefore, will be used to test the

hypothesis that damaging the inner ear may increase neural stem cell migration toward the

injured cells.

The aim of this chapter was to investigate the adaptation of the whole

cochleovestibular structure, harvested from P1 mouse pups, for in vitro analysis of factors

critical towards optimal transfer of stem cells to the inner ear. Firstly, the duration of explant

survival will be assayed. Secondly, two different methods of cell introduction into the

explants will be studied. Thirdly, the optimal confluence percentage and cell count will be

determined for c17.2 cell introduction into the explant. Then the effect, if any, of ototoxins

upon explant survival will be evaluated. Lastly, the effect, if any, of ototoxin pre-treatment

and neurotrophin co- treatment on c17.2 NSC survival within the explant will be tested.

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3.2 Results

3.2.1 Cochleovestibular Explants Survive Up to 6.25 Days

All explants were successfully harvested from the mice and architectural integrity of

the otic capsule as well as the cochleovestibular membranous spaces were maintained before

the duration survival assay started.

Explants fixed and processed from day 0.25 to 6.25, all had intact cochleovestibular

architecture and epithelium (Figure 2 – see page 69). The four explants maintained for

longer than 6.25 days, all displayed loss of cells and damaged cellular architecture (Figure 2

– see page 69), suggesting cell death within these explants. Subsequent experiments were all

therefore kept below a 6.25 day duration.

3.2.2 Microinjection Is a More Effective Method of Stem Cell Delivery into the Explants

To test the ability of c17.2 cells to be delivered into all areas of the cochleovestibular

explant, two different delivery modalities were assessed, direct application to an open

cochlea and microinjection into a cochleostomy site.

Direct application of c17.2 cells resulted in low numbers of cells within the explant.

Cells tended to remain near the cut/ damaged edge of the explant with only a small

percentage 0.5% (range 0 – 0.9%, standard deviation 0.2) migrating within the undisturbed

central part of the explant (Figure 3 – see page 70). Cells directly applied to the

69

Figure 2A

Figure 2B

70

Figure 3A

71

Figure 3B

Figure 3C

72

exposed basal spiral ganglion remained, divided and survived on top of and within the

basal spiral ganglion structure (Figures 3B and 3C – see 71).

Microinjection into a cochleostomy was a statistically more effective method (p<0.05

- 0.005) for introducing higher numbers of cells into all areas of the cochlea and vestibule

than direct application of cells onto open cochleostomy and vestibulotomy sites (Table 4 –

see page 73). The microinjection method allowed cells to be introduced into the mid and

apical portions of the cochlea, as well as the vestibule; this was not the case with direct

application of cells onto the cochleostomy or open vestibule. As would be expected, using

Kruskall Wallis analysis of ranks of the mean cell counts for each of the six animal groups

compared, there were no statistically significant differences in basal spiral ganglion cell

counts when comparing direct application to microinjection.

3.2.3 C17.2 Cells Are Optimised for Delivery at a Confluence of 70% & Concentrations of 1

Million Cells / mL

To investigate the effect of cell confluence percentage at the time of trypsinization on

c17.2 survival within explants, cells at 50, 70 and 100% confluence were trypsinized,

harvested and introduced into the inner ear explant via microinjection.

Maximal c17.2 cell survival numbers within all areas of the cochleovestibular explant

occurred at a 70% confluence rate (p<0.01 - 0.05). C17.2 cells grown to either 50% or

100% confluence demonstrated reduced survival in all areas of the cochlea and vestibule,

with no statistically significant difference seen between 50 % and 100% when compared

to 70% confluence (Table 5 – see page 73).

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Table 4: Cell Introduction Method Assay

Table 4 is comparing cell survival within cochleovestibular explants using different methods of cell

introduction. The rows represent location of cell survival following explant fixation and processing. The

columns represent the mean cell count in the region using the particular technique of cell introduction to be

studied (from a total of 6 explants) and the p value comparing the two techniques. The figures in

parentheses represent standard deviations of the mean.

ND: No statistically significant difference noted. Table 5: C17.2 Cell Confluence Assay

Table 5 is comparing cell survival within cochleovestibular explants when c17.2 cells were trypsinized at

various percentages of cell confluence and microinjected into the explants. The rows represent location of

cell survival following explant fixation and processing. The columns represent the mean cell count in the

region (from a total of 6 explants). The figure in brackets is the standard deviation of the mean. The p

values in parentheses represent a comparison between the respective columns and the 70% column,

illustrating statistically significantly lower c17.2 cell survival numbers in the explant with 50% and 100%

confluence rates.

ND: No statistically significant difference noted.

PBS Control

Direct Application

Microinjection P Value

Vestibule 0 0 103 <0.005

Apical Cochlea

0 0 5 (1) <0.05

Mid Cochlea 0 0 48 (8) <0.005

Basal Cochlea

0 15(2) 64 (8) <0.05

Basal SGN 0 913 (58) 1053(73) ND

PBS Control

50.00% 70.00% 100.00%

Vestibule 0 53 [11] (<0.01) 105[13] 42 [8](<0.05) Apical

Cochlea 0 5 [1](ND) 0 0 (ND)

Mid Cochlea 0 18 [3] (ND) 21 [6] 4 [2](<0.05) Basal

Cochlea 0 34 [7] (<0.05) 69 [8] 21 [4] (<0.05)

Basal SGN 0 653 [37] (<0.05) 1008 [66] 450 [68] (<0.05)

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Table 6: C17.2 Cell Count Assay

Table 6 is comparing cell survival within cochleovestibular explants when c17.2 cells were

microinjected at various concentrations into the explants. The rows represent location of cell

survival following explant fixation and processing. The columns represent the mean cell count in

the region at the particular cell concentration being studied (from a total of 6 explants). The figure

in brackets is the standard deviation of the mean. The p values in parentheses represent a

comparison between the respective columns and the 1 million cells/ mL column, illustrating

statistically significantly lower c17.2 cell survival numbers in the explant with 500,000 cells/ mL

and 2,000,000 cells/ mL concentrations.

ND: No statistically significant difference noted. PBS is a negative control.

PBS Control

500,000 1,000,000 2,000,000

Vestibule 0 79 [7] (<0.05) 113 [8] 85 [4] (<0.05) Apical

Cochlea 0 0 (ND) 0 0 (ND)

Mid Cochlea 0 9 [1] (<0.05) 24 [3] 11 [2] (<0.05) Basal

Cochlea 0 46 [9] (<0.05) 77 [10] 19 [5] (<0.05)

Basal SGN 0 502 [67] (<0.05) 1126 [108] 616 [50] (<0.05)

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Figure 4A

Figure 4B

76

Once an optimal confluence rate was established, the optimal cell concentration for

microinjection was assayed. One million cells / mL was the optimal cell concentration with

maximal survival for microinjection of c17.2 cells into the explants (p<0.05)(Table 6 – page

74). With respect to areas of maximal survival, c17.2 cell survival was optimal in the basal

spiral ganglion followed by the vestibule and then the basal turn of the cochlea. C17.2 cell

survival in all areas of the cochlea and vestibule diminished with the lower (1 x 105 cells/

mL) and higher (2 x 106 cells/ mL) concentrations (p<0.05). Therefore, all subsequent

experiments using the c17.2 NSC were performed with cells trypsinized at a confluence rate

of 70% and a concentration of 1 x 106 cells/ mL.

3.2.4 Ototoxins Damage the Organ of Corti in Explants

All explants pre incubated with a concentration of Gentamicin 0.1 and 1 mM as well

as Cisplatin 50 mM showed evidence of organ of Corti injury when compared to PBS

incubated negative control incubated explants (Figure 4 – see page 75). The Gentamicin

concentration of 10 mM and the Cisplatin concentration of 200 mM, however, appeared not

only to destroy the organ of Corti, but the whole cochleovestibular explant membranous

architecture, when compared to the lower concentrations and negative control explants. Only

the cartilaginous otic capsule remained intact after application of these higher concentrations.

For the pre-incubation experiments with c17.2 cells, therefore, only Gentamicin

concentrations of 0.1 mM and 1 mM as well as Cisplatin concentrations of 50 mM were

used.

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3.2.5 Gentamicin and Cisplatin Improve C17.2 Cell Survival within Explants

As mentioned in the introduction (section 3.1), c17.2 cells have been shown to have a

tropism toward damaged neurological tissue. To evaluate the effect of hair cell damage, if

any, on c17.2 cell survival in the explants, varying concentrations of ototoxins, listed

previously, that were demonstrated to have significant effects on the organ of Corti structure,

were used to pre-treat explants.

When explants were pre-treated for 24 hours with gentamicin at concentrations of

both 0.1 mM and 1 mM, enhanced c17.2 cell survival was observed in both the vestibule and

basal spiral ganglion, when compared to control microinjections with no ototoxins (p<0.05)

(see table 7 – page 80).

Cisplatin at a concentration of 50 mM also had a significant effect on increasing cell

survival numbers, when compared to control PBS microinjections (Figure 5A & B – see page

78). With 24 hour pre-incubation of Cisplatin 50 mM, c17.2 cells were seen in statistically

significantly greater numbers in all three portions of the cochlea (p<0.05) when compared to

controls (Figure 5C – see page 79).

There was a trend towards an increased number of cells in all three compartments of

the cochlea with cisplatin compared to gentamicin, but this did not reach statistical

significance (p<0.09). Furthermore, the addition of gentamicin at 0.1 mM to cisplatin 50

mM did not potentiate the individual effects, in fact the two ototoxins together, strongly

reduced c17.2 cell survival (p<0.001) (Table 7 – see page 80).

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Figure 5A

Figure 5B

79

Figure 5C

80

Table 7: The Effect of Ototoxins upon C17.2 Survival in Explants

Table 7 is comparing cell survival within cochleovestibular explants when c17.2 cells were microinjected into explants pre-treated with various concentrations of ototoxins. The rows represent location of cell survival following explant fixation and processing. The columns represent the mean cell count in the region (from a total of 6 explants) at that specific ototoxin concentration as well as various comparative p values. The figure in brackets is the standard deviation of the mean. The parentheses in the columns “Gentamicin 0.1 mM”, “Gentamicin 1 mM” and “Cisplatin 50 mM” represent p values when comparing these ototoxins to the “NC cells no ototoxic” column. The last column in the table lists the p value when comparing the cell count outcomes for Gentamicin 1 mM to Cisplatin 50 mM. NC: Negative control. ND: No statistically significant difference noted.

Table 8: Neurotrophin Effect upon C17.2 Survival in Explants

NC no cells NC No GF BDNF NT3 BDNF + NT3

Vestibule 0 89 [21] 2453 [256](<0.001) 108 [20] (ND) 2739 [389] (<0.001)

Apical Cochlea 0 5 [0] 37 [4] (<0.01) 0 (ND) 25 [6] (<0.01) Mid Cochlea 0 18 [3] 175 [21] (<0.01) 25 [7] (ND) 188 [39] (<0.01)

Basal Cochlea 0 59 [7] 450 [76] (<0.01) 41 [9] (ND) 424 [91] (<0.01) Basal SGN 0 1011 [123] 2378 [315] (<0.01) 1337 [250] (<0.05) 2105 [344] (<0.01)

Table 8 is comparing cell survival within cochleovestibular explants when c17.2 cells were microinjected into explants co-treated with various concentrations of neurotrophins. The rows represent location of cell survival following explant fixation and processing. The columns represent the mean cell count in the region (from a total of 6 explants) with co-treatment of the particular neurotrophin and various comparative p values. The figure in brackets is the standard deviation of the mean. The p value in parentheses is the figure of the growth factor being studied compared to no GF. NC: Negative control. ND: No statistically significant difference noted. GF; Growth Factor. BDNF is Brain Derived Growth Factor at a concentration of 0.1 ng/ mL and NT3 is Neurotrophin 3 at a concentration of 5 ng/ mL.

NC PBS

NC cells no ototoxics

Gentamicin 0.1 mM Gentamicin 1 mM Cisplatin 50 mM

Gentamicin 0.1 mM and Cisplatin 50

mM

P Value Gent 1 mM

compared to Cisplatin 50

mM Vestibule 0 103 [10] 175 [10] (<0.05) 180 [20] (<0.05) 154 [19] (<0.05) 8 [1] ND

Apical Cochlea 0 5 [1] 0 (ND) 0 (ND) 3(ND) 0 ND Mid Cochlea 0 21 [3] 51 [3] (<0.05) 44 [9] (<0.05) 56 [10] (<0.05) 0 ND

Basal Cochlea 0 64 [10] 49 [8](ND) 62 [11] (ND) 87 [12] (ND) 0 ND Basal SGN 0 991 [34] 1608 [96](<0.05) 1455 [87] (<0.05) 1383 [375] (<0.05) 56 [5] ND

81

Figure 6A

Figure 6B

82

Figure 7A

Figure 7B

83

Figure 8A

Figure 8B

Figure 8C

84

3.2.6 Brain Derived Nerve Growth Factor (BDNF) Improves C17.2 Cell Survival

Application of BDNF 0.1 ng/ mL substantially increased cell counts in all areas of the

cochleovestibular explant, illustrated in table 8 (p<0.001)(see page 80). Cell counts in

the basal spiral ganglion were doubled relative to the control explants, with c17.2 cells

demonstrating an ability to migrate within the modiolus toward the organ of Corti

(Figures 6A & B – see page 81). Most notably cell counts in the vestibule and

semicircular canals were increased 20 fold relative to control groups (Figures 7A, 7B &

8A, 8B, 8C - see pages 82 & 83).

NT3 5 ng/mL appeared to have no enhancing effect on cell survival and cell counts.

Of note, NT3, when applied in combination with BDNF, did not attenuate the dramatic

enhancing effects of BDNF.

3.3 Discussion

The in vitro model developed and utilized in this study represents an efficient and

rapid means of assessing different factors that contribute towards optimal implantation of

stem cells in the cochlea and vestibule. The flat multi-layered inner ear explant that has been

conventionally used has two major limitations with regard to the study of cellular therapy.

Firstly, they represent a subsection of the whole cochlea and therefore preclude analysis of

the mode of introduction and cell migration and/or dispersal from the base to apex of the

cochlea. Secondly, the micro architecture within the cochlea duct of the conventional

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cochlea explants is significantly disturbed as a consequence of its removal from the whole

cochlea and will not reflect the in vivo structural conditions. Hence, the observed results

obtained with the conventional explant may not accurately predict the outcome in vivo.

The whole cochleovestibular explant utilized in this study was not confined by these

limitations and can therefore be used as a rapid screen for assessing factors critical for a

successful transplantation of stem cells in the inner ear. Moreover, the use of this model

enables analysis of the vestibular apparatus following any experimental manipulations within

the cochlea, a technique not previously described in the literature.

A major limitation of the in vitro model is its duration of survival in culture that

precludes extended analysis of the implanted stem cells, their fate and their effect upon the

cochlea. The duration of the cochleovestibular explant was determined to be 6.25 days by

morphological observation, similar in duration to the conventional cochlea and vestibular

explants (Zheng and Gao 1999; Zheng et al. 1998; Zheng et al. 1995). The short lifespan of

the in vitro model limits prolonged experiments, however, analysis of factors with immediate

impact upon cell survival can be readily addressed through the whole cochleovestibular

explant.

Of interest was the significantly higher number of c17.2 cells surviving in the basal

spiral ganglion region of the cochleovestibular explant relative to other regions of the

cochleovestibular explant. This region of the cochleovestibular explant is significantly more

traumatised compared to other regions, since it is amputated from the intracranial portion of

the cochlear nerve, leaving the remainder of the explant undisturbed. The effect of increased

migration has been demonstrated in other damaged neuronal tissue models (Kim et al. 2004;

Riess et al. 2002; Tang et al. 2003). A possible explanation for this phenomenon may be that

86

when neuronal tissue is damaged, it releases factors that are chemo-attractive, in an attempt

to recruit and stimulate neural stem cell movement. This scenario is less likely in a

devascularised, passively nourished in vitro model.

A preliminary application of the cochleovestibular explant was to assess the optimal

growth phase for harvesting the c17.2 cells from culture for delivery to the cochlea. The data

obtained demonstrates the improved survival rates of cells in the cochleovestibular explants

when harvested at 70% density or confluency compared to 50% and 100%. The lower

survival rate for cells harvested at 100% confluency may be attributed to the cell-cell contact

that may induce mitotic inhibition or quiescence. The greater survival numbers for cells

harvested at 70% relative to 50 % confluency most likely reflects underlying differences

between cell counts at 50 % and 70%. The optimal density or confluency at which cells are

harvested for a cultured stem cell line is unlikely to be extrapolated to primary cell culture

derived stem cells. It is most likely that each primary cell culture derived stem cell has

growth cycles that vary significantly from other cells and cell lines. Therefore, each stem

cell should be assayed independently to established optimal survival numbers within the

cochleovestibular explant.

The optimal cell concentration for maximal cell survival in the inner ear explant was

determined to be 1 million cells / mL. Increasing cell concentrations above 1 million cells /

mL resulted in cell aggregation causing occlusion of the microinjection cannula. It is also

possible that concentration exceeding one million cells/mL also taxes the nutrient capacity of

the environment in which the cells are introduced, therefore affecting their viability.

The cochleovestibular model has successfully validated the enhancing effect of

ototoxins upon stem cell survival in the inner ear. This apparently counterintuitive outcome

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is however, consistent with the well-documented increased stem cell migration and survival

within damaged neurological tissue (Abe 2000; Englund et al. 2002; Kondziolka et al. 2002;

Nishino and Borlongan 2000; Park 2000; Park et al. 2002). Furthermore, most cellular

transplantation literature for the inner ear utilizes the ototoxically damaged animal model to

improve cell survival and migration (Iguchi et al. 2004; Naito et al. 2004; Tamura et al. 2004;

Tateya et al. 2003). The molecular basis of the ototoxin induced enhancement has not yet

been determined and therefore it is unknown if the molecular pathways recruited by the

gentamicin and cisplatin are similar or distinct. Nevertheless, it can be inferred that the

cytotoxicity of the ototoxins damages the hair cells and adjacent spiral neurons, exerting a

trophic effect upon neural stem cells.

Following gentamicin pre-treatment of the explants more c17.2 cells survived in the

base of the cochlea explant when compared to the apex. Two possible explanations exist for

this finding. Firstly, injecting into the basal turn, with its greater volume and site of cell

introduction would intuitively suggest more cells could survive in this region. Secondly,

gentamicin has a differential effect on outer hair cell damage throughout the distribution of

the mammalian cochlea, with more cellular damage occurring from the base to the apex

(Lautermann et al. 2004; Schacht 1999; Wu et al. 2002). This differential effect in

gentamicin damage from base to apex could not be validated in this in vitro model.

Cisplatin is thought to cause a more profound and abrupt loss of hair cells and spiral

ganglions than gentamicin (Hinojosa et al. 2001; Hinojosa et al. 1995; Hoistad et al. 1998;

Imamura and Adams 2003; Kusunoki et al. 2004; van Ruijven et al. 2004). Despite this

greater toxicity, there was a trend toward, but not reaching statistical significance, of greater

stem cell survival in the cisplatin relative to gentamicin treated group. If the mechanism by

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which the two ototoxins enhance stem cell survival is similar, then the increased severity of

damage inflicted upon the model may translate into an increased neural stem cell survival.

Our data, however, suggests that increased severity by itself may not be the sole explanation

for the higher stem cell survival rate associated with the cisplatin treatment. Thus,

gentamicin concentration beyond 1 mM yielded a decreased survival rate of the NSC.

A caveat to the cochleovestibular explant model’s usefulness in determining the

effects of ototoxin administration and the ease with which these data can be transposed to the

in vivo murine model, is the use of P0 to P2 mouse pups and the efficacy of gentamicin and

cisplatin in damaging these pup structures. Substantial literature exists suggesting that

murine pups are sensitive to the effects of ototoxins (Anniko et al. 1982; Nordemar and

Anniko 1983; Zheng and Gao 1999) and our data confirms this finding. Adult mice,

however, compared to their adult counterparts have long thought to be relatively resistant to

the effects of ototoxins at least (Forge and Li 2000; Wu et al. 2001). A new protocol that

reliably damages adult mice inner hair cells using round window application of

aminoglycosides, has been published (Heydt et al. 2004). Future translation of the in vitro

ototoxin work to the in vivo setting in mice would either, require the use of this new protocol

or exclusive use of cisplatin as the insulting agent.

The enhancing effect of stem cell survival in the inner ear was also observed in

explants treated with BDNF. BDNF and NT3 are particularly important in inner ear afferent

and efferent innervation and subsequent hair cell differentiation (Avila et al. 1993; Coppola

et al. 2001; Despres and Romand 1994; Pirvola et al. 1992; Schecterson and Bothwell 1994;

Wheeler et al. 1994). Mouse knockout studies have suggested that BDNF and NT3 play a

major role in neuronal survival as well as their migration and synaptogenesis when the ear is

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being formed (Fritzsch et al. 2002; Fritzsch et al. 1999). Thus, we hypothesized that addition

of these neurotrophic factors may affect stem cell survival in the cochlea and vestibule. The

data obtained suggests that there is a significant trophic effect of BDNF upon c17.2 cells.

The c17.2 cell itself produces several intrinsic neurotrophins at high levels including Glial

derived nerve growth factor, BDNF and Nerve growth factor (NGF) but importantly not NT3

(Niles et al. 2004). It is possible that local c17.2 TrkB receptor up regulation and cell

division is activated with exogenous BDNF application. The lack of NT3 production by

c17.2 cells also suggests that these cells may lack TrkC receptors, providing a possible

explanation as to why they failed to have an increased survival rate with exogenous NT3

application except for in the region of the spiral ganglion.

Our data suggests that c17.2 cells tend to localise to the vestibule and SGN area.

Why the c17.2 cells have a tropism toward these areas and increased growth following co-

treatment with BDNF levels awaits further study. Of note, however, is that ototoxin

application to vestibular epithelium, causes an up-regulation of TrkB receptors and BDNF

production following ototoxic damage (Popper et al. 1999), creating a scenario analogous to

external application of BDNF. The similar enhancing effect of the neurotrophins and the

ototoxins upon survival of the stem cells in the inner ear, raises the possibility of common

effector molecule(s) that are generated through their diametrically opposite mechanism of

action.

The major advantage of the cochleovestibular explant is the ability to rapidly assay

various cell types, growth factors and conditions that may affect NSC survival and growth in

the inner ear. Beyond work with primary NSCs, the model may be used to evaluate

embryonic stem cells, other progenitor cells, as well as immunophenotypic differentiation

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analysis to determine fates of the implanted cells. The model and the accompanying data

provide a useful foundation in the development of stem cell transfer technology for the inner

ear.

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Chapter 4 Olfactory Progenitor Cell Transplantation into

Mammalian Inner Ears

4.1 Introduction

The success or failure of translational research in cellular therapy of the inner ear will

depend considerably upon the cell graft employed. Current graft sources rely on harvesting

either post mortem central nervous system or embryonic stem cells. Within the CNS, the

subgranular zone of the dentate gyrus and the cells of the subventricular zone of the caudate

nucleus represent the two most extensively characterized regions in which neuronal

regeneration and hence the presence of NSCs have been identified (Gage 2000; Gage 2002).

Neural regeneration has also been identified in three other peripheral regions including the

retina, spinal cord and olfactory epithelium (Boulton and Albon 2004; Calof et al. 2002;

Gage 2000; Othman et al. 2003).

Olfactory epithelium is a particularly attractive option as a potential graft source for

the ear. The olfactory epithelium is a unique junction between the peripheral and central

nervous system that contains neural progenitor cells (Calof et al. 2002; Calof et al. 1998;

Cunningham et al. 1999; Hahn et al. 2005; Khan 2003; Murrell et al. 2005). Olfactory

neurons are replenished by differentiation from progenitor or stem cells residing from as yet

92

unidentified cells within the basal layer of the epithelium see section 1.10.2 (Carter et al.

2004; Chen et al. 2004; Hahn et al. 2005; Mackay-Sim and Kittel 1991; Murrell et al. 2005).

Neural stem or progenitor cells from the olfactory epithelium offer three major

advantages over other sources of NSCs. Firstly, olfactory epithelium is the only source of

progenitor cells that has been successfully harvested from an adult human with little

morbidity, therefore making clinical application a more realistic endeavour (Murrell et al.

2005; Zhang et al. 2004). Secondly, most NSC graft sources to date rely upon post mortem

harvesting of cerebral tissue, subsequent isolation of NSC and then either homologous or

xenografted implantation of the cells into the host animal. Isolating OPCs from a human

patient could theoretically make autologous transplantation feasible. Autologous

transplantation would eliminate host versus graft reaction and/or rejection as well as the need

for immuno-suppression. Lastly, OPCs resemble hair cells in a few ways. Both sets of

sensory cells share some morphological similarities with cilia projecting from their apical

ends and basal afferent/ efferent connections. Olfactory neurons also share the same

neuroectodermal lineage as hair cells of the inner ear and therefore share many protein

markers that are expressed during development and by the mature cell (Morest and Cotanche

2004; Rubel and Fritzsch 2002). Specifically in early neural crest differentiation, both otic

vesicle epithelium and developing olfactory epithelium rely upon the notch signalling

pathway and calcium second messenger signalling systems to develop (Bryant et al. 2002;

Cau et al. 2002; Doi et al. 2004; Fekete et al. 1997; Fekete and Wu 2002). Unique common

lineage markers such as Myosin VIIa and Sox 2 also exist between the epithelia (Beites et al.

2005; Kawauchi et al. 2004; Kiernan et al. 2005; Sahly et al. 1997; Wolfrum et al. 1998).

Furthermore, both sets of epithelia during development at least, have supporting and sensory

93

cells that are derived from bipotential progenitor cells that require a proneural gene (either

Mash 1 or Math1) for commitment to neuronal differentiation. Taking all of these data

collectively, an argument exists that OPCs could potentially be more capable of integration

within otic derived epithelium than other graftable cells.

Harvesting of the olfactory epithelium and extraction of olfactory stem/ progenitor

cells has been documented in the rat (Cunningham et al. 1999; Khan 2003). The aim of this

chapter was to apply the techniques used on rats to isolate OPCs from ubiquitously

expressing GFP mouse olfactory epithelia and introduce these cells into mouse inner ears

both in vitro and in vivo. A major problem associated with transplantation studies is the

identification of the graft cells within the host tissue following transplantation. By isolation

of OPCs from transgenic mice that ubiquitously express GFP, including within the olfactory

epithelium and particularly within the OPCs (Okabe et al. 1997; Othman et al. 2003), the

GFP should serve as an easily identifiable marker protein for assessing the identity and the

fate of the transplanted cells within the cochlea.

4.2 Results

4.2.1 Olfactory Spheres with Nestin Positive Cells Can Be Isolated from the Mouse Olfactory

Epithelium

Size fractionated olfactory cells harvested from the nasal epithelia of the enhanced GFP

cDNA transgenic mice were maintained for three generations in culture medium with

demonstration of self-replication and no change in morphological phenotypic appearance. The

94

Figure 9A

Figure 9B

95

plated single cells grew into clusters or balls of cells, which could repeatably be triturated

and dissociated into single cells, replated and regrown. This finding provided some

morphological evidence for self replication, which has been previously identified in the

rat model (Khan 2003). Nestin immunoreactivity was confirmed negative with a glial

cell line and positive with the c17.2 NSC (Figure 9A & 9B – see page 94). Nestin

immunoreactivity was checked with a sample of three spheres from each generation. All

ONS derived cells expressed the GFP marker protein. The olfactory spheres contained a

subset of cells that expressed Nestin. The Nestin positive subset was identified using

anti-Nestin antibodies and Texas Red labelled secondary antibodies (Figure 9C & 9D –

see page 97). The Nestin-positive subset of the size-fractionated cells was determined to

be 5 - 7% (range 0 – 12%, standard deviation 2.2) in two representative samples counted

from three separate generations. This sample was referred to as the OPC population.

4.2.3 Differentiation of Olfactory Spheres into ß tubulin and GFAP Positive Cells

Two days after withdrawal of EGF and FGF-2 from the growth media, the mouse

olfactory spheres developed small, elongated cells associated with the olfactory spheres.

The new morphologically distinct cells were always associated with the olfactory spheres

and were either suspended off the periphery of the sphere or attached to the bottom of the

dish. The new cells numbered between 2 and 13 per sphere with a mean of five from a

representative sample of six spheres.

96

ß tubulin protein (neuronal) and Glial Fibrillary Acid Protein (glial)

immunoreactivity was compared between undifferentiated and differentiated spheres

(Table 9 – see page 99). The differentiated spheres were negative for ß tubulin

immunoreactivity but the morphologically distinct cells attached to the sphere were

positive for ß tubulin in 24% of cells (range 9 – 34%, standard deviation 5.1%) of six

sample spheres counted. The differentiated spheres and cells on the periphery of the

sphere were positive for GFAP (a marker of glial cells) in 26% of cells (range 18 to 33%,

standard deviation 3.8%). Of note once growth factors were withdrawn and mouse

olfactory spheres allowed to differentiate all the sample spheres lost their Nestin

immunoreactivity. Undifferentiated spheres were Nestin positive 5 -7% and ß tubulin/

GFAP negative in all 6 sample spheres counted.

Having demonstrated three key features of progenitor status in the olfactory

neurospheres, the ONS will be referred to, in the remainder of the work, as containing

OPCs.

4.2.4 Olfactory Neurosphere and Olfactory Progenitor Derived Cells Survived Poorly in

Cochleovestibular Explants

Cochleovestibular explants microinjected with ONS cells and then grown and maintained

in cochlea explant media showed no survival of either ONS derived cells. When the ONS injected

explants were grown in OPC media they showed poor maintenance of explant cellular architecture

(Figure 10 – see page 98). Both cochlea and vestibular epithelium and support structures

97

Figure 9C

Figure 9D

98

Figure 10

99

Table 9: Staining Characteristics of Differentiated and Undifferentiated Mouse Olfactory Neurospheres

ß tubulin positive

GFAP positive

Undifferentiated Spheres 0 0

Differentiated Spheres 24% (4.5%) 26% (3.8%)

Glial Cell Line 0 88% (5%)

C17.2 Cell Line 97% (10%) 5% (1%)

Table 9 compares ß tubulin and GFAP immunoreactivity in undifferentiated and

differentiated spheres. Glial and neural stem cell lines were used as negative and positive

controls, respectively. The percentages were taken from a mean of six spheres. The

numbers in parentheses represent standard deviations of the mean.

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showed cell lysis and destruction. However, ONS derived cells, identified by GFP

fluorescence were repeatably observed at 2 days in the region of the spiral ganglion

before and after sectioning (Figure 10 – see page 98). These ONS/OPC derived cells

tended to survive in the region of the spiral ganglion within these explants when observed

under microscopy. Of note, all the microinjected explants maintained and fixed at time

points beyond 2 days did not survive and showed no ONS derived cells within their

architecture.

4.2.3 ONS and OPC Derived Cells Survive Robustly In Vivo

The ONS/ OPCs were assessed for their viability in the mouse cochlea in vivo

following microinjection into the basal turn of the cochlea via the round window.

Control animals comprised of PBS and c17.2 microinjected cochleae. The implanted

cells were identified by Lac Z staining (c17.2 NSC), β galactosidase immunofluorescence

(c17.2 NSC), GFP fluorescence (ONS/ ONS derived) and nestin immunoreactivity

(OPC). Both c17.2 and ONS/ OPC cells survived in the mature mammalian cochlea at

four weeks.

ONS and OPC derived cells survived in statistically significant greater numbers in the

cochlea compared to c17.2 NSCs (p<0.001 - 0.01). Different migration and/or localization patterns

were displayed by the c17.2 NSC and the ONS/ OPC derived cells following their implantation in

the mouse cochlea (Table 10 – see page 101). C17.2 NSCs survived in the scala tympani throughout

the cochlea, compared to PBS controls (Figure 11A & 11B – see page 102). ONS and OPC derived

cells, however, survived and migrated throughout all compartments of the cochlea in all animals

(Figure 11C, 12A & 12B – see pages 102,103 & 104). The statistically greatest regions of

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Table 10: C17.2 and ONS/ OPC Derived Cells in the Cochlea and Vestibule after In Vivo Microinjection

Table 10 is comparing cell survival within the in vivo model when c17.2 and ONS cells

were microinjected into mice. The rows represent location of cell survival following

cochlea and vestibule fixation and processing. The columns represent the mean cell

count in the region (from a total of 6 animals) after the specific cell types to be studied

were introduced. The value in brackets is the standard deviation of the mean. The p

values in parentheses represent comparative values between the Nestin positive (“OPC”)/

ONS derived (GFP positive) cells and c17.2 cells.

NC: Negative control. ND: No statistically significant difference noted.

NC no cells

Nestin Positive (“OPC”) ONS/ ONS Derived C17.2

Vestibule 0 684 [71] (<0.001) 2330 [315] (<0.001) 0

Apical Cochlea 0 11 [2] (<0.001) 45 [9] (<0.01) 0 Mid Cochlea 0 48 [10] (<0.05) 181 [37] (<0.01) 13

Basal Cochlea 0 92 [20] (ND) 387 [55] (<0.01) 76 Basal SGN 0 101 [35] (<0.01) 363 [86] (<0.01) 0

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Figure 11A Figure 11B

Figure 11C

103

Figure 12A

104

Figure 12B

105

Difference between OPC derived and c17.2 cells were in the vestibule, spiral

ganglion and apical cochlea (p<0.001 – p<0.01). In all three of these regions, no c17.2

cells were found after 4 weeks.

ONS/ OPC derived cells were also seen in large numbers in the endolymphatic

space. The ONS/ OPC derived cells overcrowded the scala media, distorting Reissner’s

membrane, growing up to and around the organ of Corti in four of six animals (66%).

The c17.2 cells in contrast remained in the perilymphatic space in which they were

introduced, none were found in the endolymphatic space. Of note, ONS cells were found

not just at and near the site of introduction in the basal turn of scala tympani, but also at

distant sites such as the apical cochlea and vestibule. Approximately 25% (range 18–

32%, standard deviation 5.3) of ONS/ OPC derived cells were positive for Nestin

staining. There was a trend toward more Nestin positive OPC derived cells in the

vestibule (29%) compared to the rest of the inner ear compartments, although this did not

reach statistical significance (p = 0.1).

The robust and rapid growth by the ONS and OPC derived cells resulted in

“tumour-like” balls in the endosteal/ epithelial lining of the perilymphatic space,

encroaching on the lumen of the fluid filled chamber in several of the implanted animals

(Figures 13A & 13B – see page 106). Nestin staining confirmed the presence of

significant numbers of OPC derived cells within four such balls; mean 18% (range 6 –

24%, standard deviation 3.3).

Despite difficulty with fixation and sectioning, ONS and OPC derived cells were identified

in vast numbers in the vestibules of 75% of implanted animals. Half of the OPC implanted

animals with cells in the vestibule (38%) displayed head tilting and circling gait towards the

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Figure 13A

Figure 13B

Figure 13C

107

Figure 14A

Figure 14B

108

direction of the operated ear. Inner ear histopathology of the animals with the abnormal

gait revealed a dense presence of the ONS and OPC derived cells within the vestibule of

the implanted animals (See Figure 14A & B – see page 107). GFP positive cells

completely filled the vestibule in affected animals, suggesting a rapid and abundant

growth of the introduced cells limited only by the bony confines of the vestibule.

4.2.4 Microinjection of the In Vivo Cochlea Caused No Structural Damage and No Injected

Cells Were Found in Contralateral Inner Ears

Control animals microinjected with PBS and sacrificed at 4 weeks post procedure

showed no evidence of damage from the microinjection process, in either the ipsilateral

or contralateral inner ear. In addition, no inflammatory cells were identified at the 4-

week stage in PBS control animals.

Contralateral cochleae in mice microinjected with either c17.2 or ONS/ OPC cells

showed no evidence of implanted cell survival when animals were sacrificed at 4 weeks.

4.3 Discussion

This work has demonstrated that olfactory neurospheres which contain olfactory progenitor

cells can be harvested and maintained from the mouse model, therefore validating previously

published work in the rat model (Khan 2003). These mouse olfactory neurospheres displayed

several important features defining progenitor status including morphological self-replication,

nestin positive immunoreactivity and the ability to differentiate into morphologically different cell

shapes with staining characteristics of neuronal (ß tubulin) and glial lineage (GFAP). Based on

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these data therefore, it could be inferred that the isolated mouse olfactory neurospheres contain

olfactory progenitor cells. Putative olfactory stem cells are likely to reside in these olfactory

neurospheres, although as yet, just as in the rat model no clearly defined markers exist to isolate

the olfactory neural stem cell within this population (Khan 2003).

A relevant finding in these data was the lack of successful co-culturing of the OPCs

and cochleovestibular explants (see section 4.2.2). With repeated attempts at the explant and

OPC co-culture assay, it was observed that OPCs required very specific culture conditions to

grow and survive. Any variation of the OPC growth protocol tended to cause few cells to

survive. When grown in optimal OPC conditions, the explant architecture was significantly

disrupted within 12 hours. The explant media and OPC neurobasal media differed most

significantly in the addition of L-Glutamine, EGF and FGF-2 into the OPC media. Since the

media were purchased from the same company with otherwise similar additives, it may be

inferred that it was one of these three supplements that was involved in the death of explant

cells. This finding highlights the considerable hurdles involved in finding a suitable in vitro

model for testing primary neural stem/ progenitor cell growth in the inner ear system when

compared to the robust neural stem cell line used to accumulate most of the in vitro data.

This work has demonstrated that mouse ONS/ OPC derived cells have the potential for

transplantation into the inner ear and the work has also revealed their potential destructive effects.

ONS and OPC derived cells appear to have a significantly greater migratory and growth capacity

than c17.2 neural stem cells in the inner ear. C17.2 cells remained in the perilymphatic space and

did not manage to grow in either the spiral ganglion or vestibule. ONS and OPC derived cells in

contrast, often filled the whole cochleovestibular structure and appeared to migrate into the spiral

ganglion, possibly through osseous perforations in the canal of Rosenthal or in the lining of the

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perilymphatic space (Shepherd and Colreavy 2004). Explanation for the extensive migration,

survival and growth of ONS/ OPC derived cells lies in either the “environmental niche” into which

they are transplanted, or in the cell itself.

The inner ear contains neurotrophins, including Neurotrophin 3, BDNF and FGF which are

prominent in the development of the inner ear, important in maintenance of inner ear neural

connectivity and upregulated during injury (Bowers et al. 2002; Coppola et al. 2001; Ernfors et al.

1996; Ernfors et al. 1995; Fritzsch et al. 1997a; Gao 1998; Gao 1999). Neurotrophin 3 has

recently been demonstrated to enhance the proliferation of olfactory ensheathing cells. It is

plausible that a similar phenomenon of Neurotrophin 3 environmental influence may explain OPC

behaviour in our study. A caveat to this statement, is that these olfactory ensheathing cultures may

be contaminated (Bianco et al. 2004). A further neurotrophic explanation may lie in the presence

of BDNF and up regulation of TrkB receptors in the injected animals which affects OPCs, much

like what has been described following ototoxic insult to the ear (Popper et al. 1999). Although no

ototoxin was applied with these in vivo experiments, certainly some form of inflammatory injury

may have occurred with the introduction of the OPCs, causing an up-regulation of Trk receptors

and an increase in BDNF production. Further support to this theory lies in the fact that BDNF is

preferentially located in the vestibule and the OPC cells showed particular tropism toward the

vestibule.

It has been estimated that in vivo NSCs make up ~1–2% of all cells in the

germinal region of the adult brain and 1 in 2500 cells of olfactory neuronal in vitro

cultures (Gage 1998; Gage 2000). Calof’s group, have estimated that the olfactory

neuronal stem cells in olfactory in vitro cultures occur at a rate of approximately 1 in

2500 cells (Calof et al. 2002; Calof et al. 1998). The percentage of Nestin positive cells

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in our final filtrate before microinjection was 4% (range 0 – 12%), which is higher than

published literature (Calof et al. 2002; Calof et al. 1998; Chen et al. 2004; Cunningham et

al. 1999). Although the comparison between these two numbers is not accurate as they

represent different cultures, an alternative explanation for this higher than expected

number could also be that filtering out larger cells of the olfactory epithelium,

concentrates the pluripotent cells in our cell suspension. These findings would be

consistent with the currently held understanding that the progenitor or stem cell of the

olfactory epithelium resides in the basal cell layer, which contains physically smaller

cells when compared to the other layers of the olfactory epithelium. Of even more

interest was the relatively high percentage of Nestin positive cells identified in mice ears

four weeks following injection. Most regions of the inner ear had at least a 20%

component of cells fluorescing indicating an ONS/ OPC derived origin, with the

vestibule approaching almost 30% of the cells. An inference from the high Nestin

positivity of ONS cells introduced into the inner ear is that the OPCs themselves are

being switched on to divide aggressively and perhaps this phenomenon is occurring

preferentially in certain areas of the inner ear. Literature suggests that olfactory

ensheathing cells have a high Nestin immunoreactivity and certainly differentiation of

ONS/ OPC into their glial lineage could also explain this high rate of Nestin

immunoreactivity (Doyle et al. 2001).

A valid criticism of this work is the contrasting use of neural stem cell lines compared to

primary neural stem cell cultures in the experiments. An expected result would be considerably

more NSC growth in the inner ear of the c17.2 stem cell line compared to the primary culture. The

results contradicted this finding with c17.2 cells behaving more like primary cell cultures in that

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cell numbers found after 4 weeks were similar to primary NSC implantation (Hu et al. 2005;

Iguchi et al. 2003; Naito et al. 2004). A genetic mismatch reaction between different mice breeds

is a possible explanation for the low numbers of c17.2 cells. However, this finding most

significantly emphasises the unusual finding of such high numbers of ONS/ OPC derived cells. A

better control neural stem cell would no doubt be a primary cell culture derived cell compared to

the cell line used in this thesis.

These data are the first description of the potential destructive effects of stem cell

introduction into the inner ear. OPCs introduced into the ear created “tumour-like” balls in the

walls of the perilymphatic space, completely occluded all three lumens of the cochlea and filled

the vestibule, causing circling an observable phenomenon of unilateral vestibulopathy. Several

overlapping scenarios may explain this effect. Firstly, OPCs may be triggered to switch on to

rapid, aggressive growth when in the environmental niche of the inner ear, analogous to oncogene

activation. Secondly, inflammation, from an infectious initiator, trauma, or a genetic mismatch

(such as a host vs. graft phenomenon) may have caused the extensive cellular infiltration and

destruction. Infectious responses causing the damage are less likely as the inner ear is an

immunoprivileged area, similar to the CSF space and therefore relatively protected from

inflammatory response. Lack of tissue damage with PBS microinjected control animals at four

weeks, argues against a traumatic inflammatory response being responsible for the destructive

outcomes. It could be conceivable, however, that an acute inflammatory response was triggered

by the microinjection of cells and the inner ear healed from this response before the control

animals were sacrificed at four weeks. If this scenario was used to explain the results, then

certainly an acute inflammatory response and the associated release of mediators and recruitment

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of inflammatory cells could explain the OPC reaction and overgrowth. Genetic mismatch may

also explain the cellular reaction and will be addressed with future experimentation.

Two reports to date in the literature have described tissue responses to introduced cells in

the inner ear. In the first report, dorsal root ganglion cells were transplanted into the inner ear. In

most animals with surviving neuronal tissue either no or minimal inflammatory responses were

observed. In most of the animals with no surviving neuronal tissue, the inflammatory response

and haemorrhage were significant (Olivius et al. 2004). In the second report, embryonic stem cells

were introduced into the scala media and no significant inflammatory response was seen with any

animal (Hildebrand et al. 2005). These data concur with the numerous reports listed in Table 1

where no or minimal evidence for inflammatory response exists in the inner ear after cellular

therapy. Taken collectively these data support the hypothesis that a significant overgrowth of cells

occurs with minimal inflammation. This scenario appears to have occurred with ONS/ OPC cell

introduction. GFP fluorescence was observed in multiple sites through all of the animals often

occluding cochlear ducts, indicating significant cell survival. Certainly, an inflammatory response

may be contributing to cell overgrowth, but if this is the case, ONS and OPC derived cells are still

surviving in substantial numbers amongst a non fluorescing inflammatory response four weeks

following surgery.

The demonstration of an uninjured contralateral cochlea with no cell survival

noted within the structure is the first report in the literature addressing the issue of cell

dissemination to the opposite ear in the literature. The presence of transgene expression

in the contralateral cochlea following adeno-associated viral administration via mini-

osmotic pump has been demonstrated (Kho et al. 2000). The authors speculated that this

expression may occur via haematogenous spread, through bone marrow of the temporal

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bone or alternatively via the CSF space. They suggested that microinjection is a

preferable technique of introduction to the continuous infusion of a mini-osmotic pump.

The finding of lack of inflammation, architectural damage and OPC cells in the

contralateral ear following OPC microinjection corroborates their suggestion and

provides some support for the safety of cellular therapy, at least for the contralateral ear.

In conclusion, this work provides the first documentation of the ability of OPCs to survive

and migrate in all compartments of the inner ear. Furthermore, the results highlight the potential

dangers of applying stem cell technology to the inner ear.

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Chapter 5 General Discussion

5.1 Core Issues in the Development of Inner Ear Biological Therapy

The work from this thesis has achieved the two aims and exposed considerably

more questions. Firstly, a novel cochleovestibular explant was successfully identified,

harvested and preserved for in vitro testing of cellular therapy agents. The explant was

tested for structural integrity and then demonstrated to show damage to the organ of Corti

with exposure to ototoxins. Subsequently, following pre-treatment with ototoxins,

gentamicin and cisplatin and co-treatment with the neurotrophin BDNF, c17.2 neural

stem cells showed increased survival within the explant.

Secondly, the ability of stem cells from the olfactory epithelium to survive in the

novel explant system and the mouse in vivo system was tested. The ONS cells were

harvested, sustained in culture and shown to stain positive for the intermediate filament

antibody nestin – a marker of progenitor cells. The ONS cells failed to survive in the

explant. However, when introduced into mice cochleae, ONS cells survived and grew in

all three compartments of the cochlea and in both perilymphatic and endolymphatic fluid

systems. Furthermore, the ONS/ OPCs survived in the SGN in these mice and in most

animals grew abundantly in the vestibule, damaging some mice vestibular systems to

such an extent that they showed head tilting and circling behaviour – a sign of peripheral

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vestibulopathy. As well as this damaging growth, ONS/ OPCs developed “tumour like”

growths in the perilymphatic walls of four mice.

Taken collectively, the results of this thesis contribute to the current body of work

on cellular therapy for the inner ear by firstly, providing a novel in vitro system which

may now be used to further study other possible graft cells. Secondly, the results

demonstrate that a novel cell graft the ONS/ OPC can survive and thrive in the inner ear.

Lastly and most importantly, the results expose significant problems that may arise from

introducing cells into the delicate system of the inner ear.

Several important hurdles in inner ear biology need to be overcome before a more

cohesive and structured approach to biological therapy can be pursued. A more thorough

understanding of the molecular mechanisms of inner ear inception, growth and

connection to the central nervous system will be an important initial step. A better

knowledge of the molecular mechanisms of cellular death and regeneration in both the

avian and mammalian model will allow future work to better define the “environmental

niche” surrounding organ of Corti and subsequent spiral ganglion neuron death.

Stem cells have been identified in the adult mammalian vestibular

neuroepithelium (Li et al. 2003a). Do these cells become active with insults to the inner

ear? If so, a better understanding of their role and the way in which they become active

will need to be elucidated. Demonstrating the presence, if any, of similar stem cells in

the adult mammalian organ of Corti would be a significant step toward identifying a

molecular target for potential therapies to treat sensorineural deafness.

For translational research of cellular therapy in the inner ear to be successful,

identifying the best cell or cells in sufficient numbers for transplantation will be critical.

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Do these cells require external or internal manipulation of their environment to best

facilitate implantation and integration into the inner ear? What is the best way and into

what place should the cells be introduced, to facilitate their transplantation into the

damaged portion of the inner ear and not affect the remainder of the functioning

anatomy? If internal environmental manipulation is required, is it one off, multiple or for

a continuous period of time and what is the best way of achieving this? This field is in its

infancy and many questions await exploration.

5.2 Research Directions for Future Work

A major flaw with respect to the in vitro model was the poor fixation of the explants. A

technique is being developed at the Garvan Institute whereby explants are perfused with

cryosection embedding media OCT using vacuum infusion. Preliminary results are establishing

far superior fixation to the passive permeation of OCT used in this thesis. Future studies will also

confirm the survival of explant cells with BrdU analysis as well as morphological integrity.

Furthermore, with future experiments we will use phalloidin and myosin VIIa staining to further

validate the visible hair cell damage caused by ototoxin administration in the in vitro model.

Future work undertaken from this thesis with the in vivo model will extend the current

findings and address several flaws. Firstly, a better identification and characterisation of the

pluripotent cell in the basal layer of the olfactory mucosa will need to be determined to refine cell

migration and differentiation. With time, better clarification of immunohistochemical markers for

olfactory neuroepithelium will help in separating the stem cell from other cells of the basal region

of the olfactory epithelium. Using Mash 1 -/- knockout models of olfactory progenitor

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overgrowth, up-regulation of two key transcription factors has helped further refine the search for

the putative olfactory stem cell (Beites et al. 2005; Calof et al. 2002). Sox 2, a homeobox

transcription regulator and RALDH3, a retinoic acid dehydrogenase show up regulation in these

rapidly dividing olfactory neuronal precursors. Fluorescence Activated Cell Sorting using Sox 2

and/ or RALDH3 may yield a more pure form of olfactory neural stem cell, more proximal in

lineage to the precursors that likely exist in the olfactory neurospheres used in this thesis.

A better candidate NSC control cell than the neonatal cerebellar derived c17.2 neural stem

cell line will be used in future experiments. Using established published protocols for isolating

neurospheres from either hippocampal or lateral ventricular tissue, the P0 to P2 day old GFP

fluorescing pups will be good candidates for positive control cells in future experimentation (Ito et

al. 2001). Olfactory/ respiratory epithelium and cerebral tissue could be harvested from the same

GFP fluorescing animals – an ideal control scenario.

The issue of possible genetic mismatch causing cellular overgrowth and

cochleovestibular destruction will be addressed in future experiments by using syngeneic

animals to Dr Okabe’s GFP mice, for transplantation. Furthermore, the animals will be

physiologically screened preoperatively to ensure normal hearing and vestibular function

using auditory brain stem response measures and behavioural testing in particular testing

righting reflex, hanging tail test and swimming patterns (Gray et al. 1988; Hunt et al.

1987; Ossenkopp et al. 1990).

In vivo study groups will then be divided into untreated and ototoxically damaged

animals. Mice will be ototoxically injured using a previously published protocol that

reliably damages hair cells using gentamicin soaked Gelfoam pledgets placed in the

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round window niche (Heydt et al. 2004). Purified OPCs will then be introduced,

survived and examined as described in the thesis.

With future experiments, animals will have auditory brain stem response and

vestibular behavioural testing during the study period (preoperatively, day 3, 7, 14 and

28) to determine if and when, there is a decline in audiological and vestibular function.

Following animal sacrifice, analysis of cell fate phenotypic outcomes will be studied

using established olfactory progenitor (Mash 1, Sox2, RADLH3), hair cell (Myosin

VIIa), spiral ganglion, neuronal (Microtubule Associated Protein 2) and glial markers

(GFAP).

To better understand the observed finding of cellular overgrowth and destruction,

immunohistochemistry will be used to analyse the sectioned cochleae and vestibules.

Immunohistochemistry will be performed to look for signs of acute and chronic

inflammation including markers of lymphocytes and macrophages e.g. anti CD45

antibody and anti L1 antibody (Wareing et al. 1999). In addition, the identification of

tumourigenic markers that suggest the expression of proteins not present on pre-

transplanted OPCs would provide some evidence as to the underlying cause for rapid cell

growth and damage. Improved analysis of the cell counts will be undertaken using point

stereological cell counting to better establish a volume density relationship of the

transplanted cells to their location. Local equipment based at the Garvan Institute will

allow better image acquisition and data analysis than what was presented in this thesis.

Finally as an extension of the in vitro findings for BDNF effects on c17.2 cells, an

assay will be performed where animals are divided into groups that receive and do not

receive concurrent neurotrophins when the ONSs and Pup GFP derived hippocampal

120

neurosphere cells are transplanted, the anatomical and physiological outcomes can be

determined. Various combinations of BDNF, NT3 and BNDF/ NT3 will be

concomitantly applied with the cells and anatomical/ physiological outcomes measured as

outlined in this thesis.

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5.3 Concluding Remarks

Work in the field of stem cell biology and technology is just in its infancy.

Substantial progress has been achieved in the identification and understanding of these

cells, which will eventually assume a place in the treatment paradigm for many diseases.

It is likely in the field of biological therapy for the inner ear, that some of the significant

findings of the past ten years, in particular with neurotrophin and gene therapy, will soon

bear fruition by clinical application. The most likely near future scenario for treatment of

sensorineural deafness will be a hybrid of electrical and biological therapy, perhaps

where either neurotrophins or genes are applied to the cochlea via a cochlear implant.

122

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