Stem cells and optogenetics: novel approaches for the treatment of focal ischemic stroke in adult mice Open Access

Mohamad, Osama (2012)

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Stroke is the fourth leading cause of death and the number one cause of disability in the adult population in the United States in 2011. Despite the economic, healthcare and social burden of stroke, therapy is still limited to one FDA approved drug (tPA), mechanical thrombolysis, supportive care and rehabilitation. Stem cell-based therapies especially human induced pluripotent stem (iPS) cells offer new hope for stroke and many other degenerative diseases. However, the mechanisms of stem cell-induced benefit after transplantation are not fully understood and the neuronal differentiation and post-injury transplantation protocols have not been optimized yet.

In this dissertation, we first tested the therapeutic potential of vector-free human induced pluripotent stem (hiPS) cells in a mouse model of focal ischemic stroke. hiPS cells differentiated into functional neurons in vitro staining positively for mature neuronal markers and firing trains of action potentials. After transplantation in stroke mice, hiPS cells survived and differentiated to neurons with no signs of tumor formation. Transplantation of hiPS cell-derived neural precursors alleviated sensorimotor deficits, increased trophic support and restored neurovascular architecture after stroke. We also developed optogenetics tools to be applied in stroke research and stem cell therapies. Channelrhodopsin-2 (ChR2) was over-expressed in cortical neurons in vitro and in vivo and in mouse iPS cell-derived neurons. Functional responses to light stimulation were recorded in vitro and in brain slices. Pre- and post-stroke light stimulation in the penumbra of ChR2 expressing mice reduced infarct volume and accelerated behavioral recovery, respectively.

Second, we developed a new protocol for the neuronal differentiation of mouse pluripotent stem cells. Culturing embryoid bodies (EBs) from D3 mouse ES cells on a rotary shaker produced smaller and more uniform neurospheres and increased the yield of neurons after the 4-/4+ differentiation protocol by 4-5 times. Terminally differentiated neurons were morphologically, functionally and immunohistochemically similar to those produced with static cultures.

Collectively, these data suggest that stem cell transplantation is a promising therapy for stroke and that optogenetics provide valuable tools for stroke and stem cell research. More work, however, is required to optimize neuronal differentiation and transplantation protocols before transitioning to human clinical trials.

Table of Contents

Chapter I. Ischemic Stroke...1 A. Stroke facts...2 1. Definition
2. Epidemiology
3. Causes B. Ischemic injury...4 1. Molecular events following ischemia
2. Inflammation C. Endogenous repair and trophic support...9 1. Neurogenesis
2. Angiogenesis D. Stroke treatment...12 1. Prevention
2. Currently available therapies
3. Cell-based therapies E. Experimental models of ischemic stroke...16 1. Focal ischemia models
2. Global ischemia models
3. Oxygen-glucose deprivation: in vitro ischemia F. Summary and conclusions...19 Chapter II. Pluripotent stem cells...20 A. Stem cells: an introduction...21 1. Embryonic stem (ES) and induced pluripotent stem (iPS) cells
2. Introduction to transplantation B. Stem cells in stroke clinical trials: an overview...24
C. Pluripotent stem cell differentiation...26 1. Mouse ES cell neuronal differentiation
2. Human iPS cell neuronal differentiation D. Routes of stem cell transplantation...30 1. Local ipsilateral transplantation
2. Contralateral transplantation
3. Vascular delivery
4. Intranasal delivery E. Strategies to enhance cell survival in vivo...34 1. Control of cell number and timing of delivery
2. Genetic manipulation
3. Preconditioning
4. Cotransplantation F. Mechanisms of stem cell induced benefit...37 1. Trophic support and attenuation of inflammation
2. Cell differentiation and integration
3. Reduction of infarct volume G. Immune response in stem cell therapy...39 1. Inflammation
2. Tumor formation
3. Immune rejection H. Summary and conclusions...45 Chapter III. Optogenetics...47 A. Historical overview and introduction to opsins...48
B. Opsin delivery...52
C. Light delivery...55
D. Optogenetics and stem cells...56
E. Summary and Conclusions...57 Chapter IV. Rationale, Aims, and Experimental Methods...58 A. Rationale and significance...59
B. Specific aims...61
C. Materials and Methods...62 Chapter V. Human iPS cells differentiate to functional neurons and improve functional and behavioral recovery after ischemic stroke...77 A. Introduction...78
B. Results...81 1. Characterization of hiPS cells cultured in serum-free and feeder-free media
2. Neural differentiation of hiPS cells
3. Human iPS cell-derived neurons exhibit functional neuronal characteristics
4. Transplantation of hiPS cell-derived neural precursors after focal cerebral ischemia in mice
5. hiPS cell-derived neural precursor transplantation enhances functional behavioral recovery after stroke
6. Local cerebral blood flow after transplantation
7. Intrinsic optical signal imaging after transplantation
8. Trophic factor expression in hiPS cell-derived neural precursors and after transplantation
9. Neurogenesis and angiogenesis after hiPS cell-derived neural precursor transplantation
10. Whisker stimulation enhances behavioral recovery after stroke but not synergistically with neural precursor transplantation C. Discussion...102 Chapter VI. Establishing Optogenetic Tools for Studying and for the Treatment of ischemic stroke...108 A. Introduction...109
B. Results...112 1. Establishing optogenetic techniques for stroke and stem cell research
2. Testing optogenetic tools in vitro: expression and function of hChR2 in mouse cortical neurons
3. Testing optogenetic tools in vitro: expression and function of hChR2 in mouse iPS cells
4. Testing optogenetic responses in ex vivo brain slices
5. Using in vivo optogenetic tools to study stroke mechanisms C. Discussion...123 Chapter VII. Highly efficient production of neurons from mouse ES and iPS cells...126 A. Introduction...127
B. Results...130 1. Rotary cultures increase neurosphere homogeneity and the number of neural precursors from mouse ES cells
2. Reduced cell death and increased proliferation in EBs and neurospheres in rotary cultures
3. Increased immature neuronal marker Tuj-1expression in day 8 neurospheres of rotary cultures
4. Rotary cultures produce neurons with similar phenotype and function as static cultures
5. Rotary cultures increase yield of NPs from mouse iPS cell differentiation and produce mature and functional neurons C. Discussion...141 Chapter VIII. Summary and conclusions...145
Chapter IX. References...148

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