Optogenetic Stimulation of Transplanted Stem Cells to Enhance Regeneration and Repair After Traumatic Brain Injury Open Access

Zhang, James (Fall 2017)

Permanent URL: https://etd.library.emory.edu/concern/etds/5x21tf41f?locale=en
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Abstract

Stem cells provide the unique opportunity to address the treatment gap for many intractable disorders, including traumatic brain injury (TBI). TBI is the leading cause of morbidity and mortality amongst children and young adults. Despite its being such a prevalent healthcare burden, there are currently no effective treatment options for TBI. While stem cell therapy has emerged as a promising candidate for treatment, it remains suboptimal. One of the primary challenges with cell therapy is the hostile transplantation environment and the glial scar, which prohibit neuritogenesis and plasticity in the peri-contusion region. To bridge this treatment gap, I genetically engineered a stable induced pluripotent stem cell (iPSC) line that expresses the fusion protein luminopsin (LMO), which is comprised of a light-emitting luciferase with a light-sensitive excitatory channelrhodopsin, to allow for control of neural activity by intranasal delivery of the luciferase substrate, coelenterazine (CTZ). I first demonstrated that photostimulation of iPS-neurons by either CTZ or laser both resulted in upregulation of activity-dependent factors BDNF and NGF within in vitro models of iPS-derived neurons, as well as enhancement of axon outgrowth and plasticity, even beyond regions of axonal inhibition, such as into CSPG droplets. Next, the neurotrophic upregulation with intranasal CTZ stimulation was corroborated in the controlled cortical impact (CCI) mouse model of TBI. Furthermore, stimulation resulted in greater axonal outgrowth and reorganization with enhanced parallelism of axon bundles. CTZ stimulation also improved endogenous neurovascular repair by promoting neovascularization and restoring local cerebral blood flow (LCBF), which counters ischemic cytotoxic edema, a key secondary effect of TBI. As a result, stimulation enhanced the neuroprotective effect of stem cell transplantation, which led to decreased contusion volumes and improved functional outcomes. Overall, I have established and characterized a next-generation stem cell strategy to augment the function and efficacy of cell therapy after transplantation by driving grafted cell activity. While this dissertation has focused on the efficacy of this combination therapy for TBI, this novel non-invasive approach provides great translational potential and sets the precedence for diverse stem cell applications and treatments.

Table of Contents

Table of Contents

Chapter 1: Traumatic Brain Injury .............................................................................. 1

1.1 Introduction: Epidemiology, subtypes, and stratification .................................... 1

1.2 Pathophysiology ................................................................................................... 2

1.3 Current Status of Treatments................................................................................ 3

1.4 Animal Models of TBI ......................................................................................... 3

1.4.1 Controlled cortical impact............................................................................. 3

1.4.2 Fluid percussion injury ................................................................................. 4

1.4.3 Weight drop .................................................................................................. 4

1.4.4 Blast model ................................................................................................... 5

1.5 Endogenous response and extent for brain repair ................................................ 6

1.6 Summary .............................................................................................................. 7

Chapter 2: Stem Cell Therapy ..................................................................................... 8

2.1 Introduction .......................................................................................................... 8

2.2 Stem Cell Types ................................................................................................... 9

2.2.1 Embryonic stem cells .................................................................................... 9

2.2.2 Induced pluripotent stem cells .................................................................... 10

2.2.3 Adult neural stem cells................................................................................ 12

2.2.4 Mesenchymal stem cells ............................................................................. 12

2.3 Neuronal Induction of Pluripotent Stem Cells ................................................... 13

2.4 Transplantation Routes ....................................................................................... 15

2.4.1 Intracranial (intracerebral or intraventricular) ............................................ 15

2.4.2 Intravenous or Intra-arterial ........................................................................ 16

2.4.3 Intranasal ..................................................................................................... 16

2.5 Timing of Transplantation .................................................................................. 17

2.6 Mechanisms Underlying Cell Therapy .............................................................. 18

2.6.1 Trophic support: neuroprotection and suppression of inflammation .......... 18

2.6.2 Stimulation of Angiogenesis ....................................................................... 20

2.6.2 Enhancement of endogenous neurovascular regeneration .......................... 22

2.6.3 Cell Replacement ........................................................................................ 23

2.7 Transplantations in Animal Models and Outcomes ........................................... 24

2.8 Clinical Trials of Stem Cell Therapy for TBI .................................................... 26

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2.9 Summary ............................................................................................................ 28

Chapter 3: Evolving Frontiers of Cell Therapy ........................................................ 29

3.1 Performance Enhancing Factors......................................................................... 30

3.2 Preconditioning Approaches .............................................................................. 32

3.3 Cell Carriers for Improved Delivery .................................................................. 33

3.4 External Stimulation for Activity-Driven Plasticity........................................... 34

3.4.1 Activity-Dependent Plasticity During Development and After Injury ....... 34

3.4.2 Stimulation Modality: Neuroprosthetics ..................................................... 36

3.4.3 Precision Stimulation by Genetic Engineering ........................................... 37

Chapter 4: Optogenetics ............................................................................................ 38

4.1 Introduction ........................................................................................................ 38

4.2 The Expanding Optogenetic Toolbox ................................................................ 39

4.3 Targeted Delivery of Opsins .............................................................................. 41

4.4 Delivery of Light ................................................................................................ 43

4.5 Luminopsins: Next-Generation Optogenetic Fusion Proteins ........................... 44

4.6 Optogenetics and Stem Cells.............................................................................. 47

Chapter 5: Rationale, Aims, and Experimental Methods.......................................... 49

5.1 Rationale and Significance ................................................................................. 49

5.2 Specific Aims ..................................................................................................... 52

5.3 Experimental Methods ....................................................................................... 54

Chapter 6: TBI Model and Neurovascular Response................................................ 76

6.1 Introduction ........................................................................................................ 76

6.2 The Controlled Cortical Impact Model .............................................................. 76

6.3 TBI-Induced Autophagy and Cell Death ........................................................... 80

6.4 Endogenous Repair Mechanisms After TBI ...................................................... 83

Chapter 7: Aim 1 – Creation and in vitro Characterization of LMO3-expression Stable iPS Cell Line .......................................................................................................... 94

7.1 Introduction ........................................................................................................ 94

7.2 Results ................................................................................................................ 96

7.2.1 Aim 1.1 – Create and validate an LMO3 plasmid containing the fused VChR1-sbGLuc transgene. ........................................................................................ 96 7.2.2 Aim 1.2 – Establish an iPS cell line that expresses LMO3, which will be functionally validated by performing in vitro luminescence and electrophysiology studies. 101

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7.2.3 Aim 1.3 – Determine neurotrophic responses to photostimulation at different stages of neural progenitor cell differentiation ......................................... 110

7.3 Discussion ........................................................................................................ 123

Chapter 8: Aim 2 – Enhancement of iPS Cell Therapy by CTZ Stimulation After TBI 125

8.1 Introduction ...................................................................................................... 126

8.2 Results .............................................................................................................. 128

8.2.1 Establishment of improved transplantation technique and evaluation of iPS-neuronal migration after transplantation .................................................................. 128

8.2.2 Aim 2.1 –Validate cell viability and differentiation and LMO3 function after transplantation of LMO-expressing iPS-NPCs in a mouse barrel cortex TBI model 133

8.2.3 Aim 2.2. – Assess effectiveness of CTZ stimulation for upregulating activity-dependent neurotrophin release and enhancing endogenous plasticity and neurovascular repair ................................................................................................ 138

8.2.4 Aim 2.3 – Compare functional recovery of TBI animals treated with combination therapy of iPS-NPC transplantation and CTZ stimulation vs. iPS-NPC transplantation alone ................................................................................................ 150

8.3 Discussion ........................................................................................................ 154

Chapter 9: Aim 3 - Enhancement of Luminopsins by Addition of Golgi Trafficking Element 159

9.1 Introduction ...................................................................................................... 160

9.2 Results .............................................................................................................. 161

9.2.1 Aim 3.1 – To create the virus containing eLMO3 with an inserted Golgi TS motif and evaluate the membrane localization and elicited photocurrents ............. 161

9.2.2 Aim 3.2 – To investigate the distribution of eLMO3 following AAV infection of the mouse S1 cortex and assess the functional differences between LMO3 and eLMO3 .................................................................................................. 165

9.2.3 Improved expression of LMO3 elicits greater behavioral responses........ 168

9.3 Discussion ........................................................................................................ 171

Chapter 10: Summary and Conclusions ................................................................ 173

Chapter 11: References ......................................................................................... 176

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