Abstract
Ischemic stroke is a leading cause of death and disability
in the United States. Treatment options are currently limited, and
currently only one FDA-approved therapy exists (tPA). Although tPA
was a great stride forward in stroke treatment, only a small
percentage of patients are eligible to receive the drug and even
smaller actually receive it. Research to date has focused on small
molecule and peptide based therapies aimed at neuroprotection:
protecting dying neurons, glia, and vascular from ischemia-media
cell death. Decades of pre-clinical studies generated exciting
results in animal models, however all have failed to translate to
successful patient care. Currently, there are no therapies
available to promote central nervous system (CNS recovery) in
stroke survivors. Cellular therapy, the use of stem cells for
stroke treatment, has blossomed in the lab in the past decade.
Induced pluripotent stem cells (iPS cells, or iPSCs) in particular
are an excellent candidate for future stroke therapies. With
multiple mechanisms of action and the possibility for not only
neuronal cell replacement but personalized medicine as well, the
use of iPSCs for stroke therapy holds great potential. Current
studies generate as proof-of-principal that iPSC transplantation
can be safe, efficacious, and can provide a source of new neurons
that integrate into host circuitry. While these studies are
exciting, they are far from ideal. Many questions remain and in its
current form, iPSC transplantation for stroke is not optimized and
remains inefficient. This dissertation examines how iPSC
transplantation may be optimized for ischemic stroke treatment. We
examined a novel intranasal transplantation route as well as a
novel genetic manipulation method (focal adhesion kinase, FAK,
overexpression we hypothesized would improve functional recovery
over current methods. Our results suggest that the novel
non-invasive intranasal transplantation may improve functional
recovery at a similar level compared to existing methods. We also
demonstrate FAK overexpression may improve some aspects of normal
neuronal development that would aid the integration of
transplantated cells. Taken together, our data suggest that
optimization of existing iPSC transplantation methods may benefit
iPSC therapy for ischemic stroke.
Table of Contents
CHAPTER 1 - ISCHEMIC STROKE 1
INTRODUCTION 2
ISCHEMIC STROKE 3
Definition and Epidemiology 3
CELLULAR AND MOLECULAR CAUSES OF ISCHEMIC INJURY 5
Glutamate-Mediated Excitotoxicity 7
Oxidative Stress 8
Neuroinflammation 9
CO-MORBID DISORDERS: POST-STROKE DEPRESSION AND AFFECTIVE DISORDERS
11
CURRENT TREATMENT OPTIONS: THE NEED FOR NEW TREATMENTS 13
TRANSLATION STROKE RESEARCH: THE SEARCH FOR NEW TRANSLATIONAL
THERAPIES 16
Neuroprotection: Two Decades of Failure 16
Thrombolytic Research: Thrombolytics and tPA 20
SUMMARY : THE NEED FOR NOVEL APPROACHES AND CELLULAR THERAPY
22
CHAPTER 2 - CELLULAR THERAPY: STEM CELLS, IPS CELLS, AND CELL
TRANSPLANTATION 24
POST-ISCHEMIC CELLULAR THERAPY: THE NEXT FRONTIER 25
CELLULAR THERAPY: SOURCES OF CELLS 25
Endogenous NPCs, Neurogenesis, and NPC Migration 26
Neurogenesis in the Sub-Granular Zone (SGZ) 27
Neurogenesis in the Subventricular Zone (SVZ) 30
Neurogenesis in the Subcallosal Zone (SCZ) 33
Exogenous Cells 34
Neural stem cells (NSCs) 35
Mesenchymal stem cells (MSCs) 38
Embryonic stem cells (ESCs) 41
Induced pluripotent stem cells (iPS cells) 44
CELLULAR THERAPY: TIMING, DELIVERY, SURVIVAL, AND OTHER
CONSIDERATIONS 47
Timing 48
Intracranial Delivery 49
Intravenous Delivery 50
Intranasal Delivery 50
Cell Survival and Tumorigenesis 51
SUMMARY: CELLULAR THERAPY AND ITS IMPLICATIONS 52
CHAPTER 3 - STRATEGIES TO IMPROVE CELLULAR THERAPY FOR ISCHEMIC
STROKE AND FOCAL ADHESION KINASE (FAK) 54
CELLULAR THERAPY: MODIFICATIONS AND LOOKING TOWARD THE FUTURE
55
Genetic Modifications: Over-expression 55
Hypoxic preconditioning 58
Combinatorial Therapies: Cellular Therapy and Environmental
Enrichment 59
FOCAL ADHESION KINASE (FAK) 60
Protein Structure and Activation 61
FAK and Cellular Adhesion 66
FAK and Cellular Migration 68
FAK and Ischemia 70
FAK and Neuritogenesis 71
SUMMARY: INCREASED FAK SIGNALING MAY IMPROVE CELL TRANSPLANTATION
72
CHAPTER 4 - RATIONALE, AIMS, AND EXPERIMENTAL METHODS 74
RATIONALE AND SIGNIFICANCE 75
SPECIFIC AIMS 80
EXPERIMENTAL METHODS 81
CHAPTER 5 - CHARACTERIZATION OF A MODIFIED MCAO STROKE MODEL
98
INTRODUCTION 99
RESULTS 102
Local Cerebral Blood Flow (LCBF) 102
TTC Staining 104
TUNEL Staining 106
DISCUSSION 108
CHAPTER 6 - MOUSE IPS CELLS: CULTURE AND NEURONAL DIFFERENTIATION
110
INTRODUCTION 111
RESULTS 112
Cell Culture Overview 112
Mouse iPS Cell Culture 114
Neuronal Differentiation Efficiency 116
Mature Neuronal Markers 119
iPS Neuron Phenotyping 121
FAK Expression 123
iPS-NPCs Express Pro-Migratory Proteins 128
Growth Factor and Cytokine Expression 131
DISCUSSION 134
CHAPTER 7 - EFFICIENT AND STABLE TRANSGENIC IPS CELL CREATION
137
INTRODUCTION 138
RESULTS 140
Inducible Expression Construct Design 140
Inducible Construct Validation 142
iPS Transfection Optimization 144
Transfection of miPSCs and Doxycycline Induction 146
Viral Induction of Mouse iPS Colonies 149
Validation of Additional Expression Constructs 152
Lentivirus-Mediated Stable Cell Creation 155
Non-Viral Constitutive Expression Vector Design 159
Non-Viral Stable Cell Creation 161
FAK-iPS Cell Creation 166
DISCUSSION 175
CHAPTER 8 - EFFECT OF FAK OVEREXPRESSION ON IPSC ATTACHMENT,
MIGRATION, AND NEURITE OUTGROWTH 179
INTRODUCTION 180
RESULTS 181
Oxygen-Glucose Deprivation 181
iPS-NPC Attachment 184
iPS-NPC Migration 187
mCherry-FAK iPS Neurospheres Show Improved Neurite Outgrowth
190
Hypoxia Preconditioning Upregulates FAK in MSCs 192
Hypoxia Preconditioning in iPS-NPCs 194
WNT-3a Increases iPS-NPC Migration 197
DISCUSSION 199
CHAPTER 9 - INTRANASAL IPSC DELIVERY IMPROVES RECOVERY FOLLOWING
FOCAL ISCHEMIA 204
INTRODUCTION 205
RESULTS 210
iNA iPS-NPCs Reach the Brain and Ischemic Core 210
iNA-iPS Delivery Increases Local Cerebral Blood Flow (LCBF)
212
iNA-iPS Transplantation Increases Bcl-2 Expression 214
No Functional Improvement in Animals That Receive iNA-iPS Therapy
216
iNA-iPS Delivery Decreases Endogenous Neurogenesis and Angiogenesis
218
No Functional Improvement in Animals That Receive Delayed iNA-iPS
Therapy 220
Delayed iNA-iPS Therapy Has No Effect on BrdU Positive Cells in the
Ischemic Penumbra. 222
iNA-iPS Therapy Reduces Anxiety-Like Behavior 224
Delayed Intranasal Transplant Improves Anxiety Like Behaviors
227
DISCUSSION 229
CHAPTER 10 - SUMMARY AND CONCLUSIONS 234
PUTATIVE UNDERLYING MECHANISMS FOR IPS-BASED AND OTHER CELLULAR
THERAPIES 237
Cell Replacement 238
Trophic Support 239
Angiogenesis 240
Modulation of Endogenous Neurogenesis 242
Immune Modulation 244
CHAPTER 11 - REFERENCES 246
About this Dissertation
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