Induced Pluripotent Stem (iPS) Cell Transplantation as a Treatment for Ischemic Stroke Público

Chau, Monica Jannie (2014)

Permanent URL: https://etd.library.emory.edu/concern/etds/qb98mf51q?locale=es
Published

Abstract

Ischemic stroke is a leading cause of death and long-term disability in the U.S.
Despite its prevalence, there is only one FDA-approved treatment for stroke, tPA (tissue
plasminogen activator) which is a thrombolytic drug limited to use within 4.5h after
stroke. Currently, no treatment exists for the regeneration of damaged brain tissue. The
adult brain undergoes endogenous regeneration, however it cannot fully repair the
damage caused by stroke. Induced pluripotent stem (iPS) cells are a novel source of stem
cells created by genetically reprogramming one's somatic cells into pluripotent cells.
Transplanting such patient-specific cells can circumvent host immune rejection as well
as circumvent the ethical limitations of obtaining pluripotent cells from human embryos.

This dissertation demonstrates the use of iPS cell transplantation as a therapeutic
in two ways: by providing replacement cells to the infarct, and by releasing trophic
factors to the injured tissue enhancing the recruitment of regenerative progenitors. We
examine the regenerative capabilities of transplanted iPSC-derived neural progenitors
(iPSC-NPCs) with and without stromal-cell-derived factor-1α (SDF-1α) upregulation.
Our overall goal is to enhance the recruitment of endogenous progenitors to the infarct.

To support the hypothesis of our first aim, we tested the transplantation of iPSC-NPCs
in a model of neonatal ischemic stroke and were able to demonstrate an enhanced
neurogenesis and angiogenesis in the peri-infarct area and increased sensorimotor
functional recovery. In our second aim, to supplement the chemoattractive factor, SDF-
1α that is endogenously expressed after stroke, we transplanted iPSC-NPCs that had
stable overexpression of SDF-1α. We demonstrated an increased endothelial progenitor
cell recruitment for angiogenesis in mice with stroke and SDF-1α iPSC-NPC
transplantation. Furthermore, transplanting iPSC-NPCs and SDF-1α iPSC-NPCs can
provide more neuronal cells to the injury and increased functional recovery after stroke.

Overall, these data suggest that iPS cells have great potential as a personalized
transplantation therapeutic for the regenerative phase of ischemic stroke. Transplanting
SDF-1α iPSC-NPCs employs a two-fold strategy to provide more neuronal cells into the
ischemic parenchyma and to enhance the recruitment of endothelial and neural progenitor
cells for regeneration.

Table of Contents

Chapter I. Ischemic stroke……………………………………………………………....1
A. Ischemic Stroke………………………………………………………………...2
1. Background……………………………...……………………………...2
2. Epidemiology…………………………………………………...……....2
3. Risk Factors…………………………………………….……………....3
4. Treatment……………………………………………….……………....4
B. Ischemic Injury Cell Death Mechanisms……………………………………....7
1. Heterogeneous Cell death……………………………………………....7
2. Necrosis………………………………………………………………...8
3. Apoptosis…………………………………………...………………....11
a. Intrinsic pathway……………………………………………....13
b. Extrinsic pathway………………………………………….......14
c. Commonality in Both Pathways: Caspase Activation………...15
4. Ionic Homeostasis …………………………………………………….15
5. Excitotoxicity………………………………………………………….17
C. Experimental Models of Stroke……………………………………………….18
1. In Vitro Models………………………………………………………..18
2. In Vivo Models………………………………………………………..19
a. Focal Ischemia Stroke Models………………………………...19
b. Global Ischemia Stroke Models ………………………………21
E. Summary………………………..……………………………………………..21
Chapter II. Endogenous Regeneration After Stroke…………………………………23
A. Rostral Migratory Stream Migration………………………………………….24
B. Inflammation and Regeneration………………………………………………25
C. SDF-1α Plays a Chemoattractive Role in Stroke……………………………..27
1. SDF-1α is a Chemokine………………………………………………27
2. SDF-1α and Ischemia…………………………………………………29
3. SDF-1α as a Therapeutic……………………………………………...31
D. Summary……………………………………………………………………...32
Chapter III. Stem Cell Transplantation As a Stroke Therapy………………………33
A. Stem Cell Types……………………………………………….……………...34
1. Mesenchymal Stem cells………………………………………………34
2. Embryonic Stem cells…………………………………………………36
3. iPS Cells……………………………………………………………….37
B. Routes of Transplantation…………………………………………………….40
1. Intracerebral Transplantation………………………………………….40
2. Intravascular Transplantation…………………………………….……40
3. Intranasal Administration……………………………………....……...42
C. Summary………………………………………………………………...........42

Chapter IV. Stem Cells and Trophic Factors…………………………………………44
A. Trophic Factors From Stem Cells Promote Regeneration……………………46
1. Trophic Factors From Stem Cells Promote Angiogenesis…………….46
2. Trophic Factors From Stem Cells Promote Neuroprotection and
Neurogenesis……………………………………..…………………...…47
B. Strategies to Enhance Trophic Factor Expression…………………………….49
1. Genetic Upregulation of Trophic Factors……………………………..49
2. Hypoxic Preconditioning……………………………………………...52
a. HIF-1α Mechanism…………………………………………...52
C. Summary……………………………………………………………………...54
Chapter V. Rationale, Aims, Experimental Methods………………………………...56
A. Rationale and Significance……………………………………………………57
B. Specific Aims…………………………………………………………………58
C. Materials and Methods………………………………………………………..59
Chapter VI. iPS Cell Transplantation Increases Regeneration and Functional
Activities in a Model of Neonatal Ischemic Stroke
….……..…………………………75
A. Introduction…………………………………………………………………...76
B. Results………………………………………………………………………...78
1. Experimental Timeline of In Vitro Differentiation and In Vivo
Transplantation…………………………………………………..………79
2. Pluripotent iPSCs Are Differentiated Down the Neural Lineage……..80
3. iPSCs Are Differentiated into Functional Mature Neurons…………...82
4. Differentiated iPSCs Express Trophic Factors…………………..……82
5. Increased Trophic Factor Expression After Transplantation….………84
6. Transplanted iPSCs Differentiate into Neuronal-like Cells……...……85
7. Transplantation Increases Regeneratio…………..………………..…..87
8. Transplantation Increases Functional Recovery………………………88
C. Discussion………………………………………………………………….…90
Chapter VII. The Role of SDF-1α Upregulation in Cell Survival and Differentiation
In Vitro
…………………………………………………………………………..95
A. Introduction…………………………………………………………………...96
B. Results…………………………………………………………….………..…98
1. GFP-SDF-1α Can Be Expressed Under Different Promoters……...…98
2. SDF-1α Can Be Stably Expressed in iPSCs……………………..……99
3. SDF-1α Expression is Maintained Throughout Stages of Neuronal
Differentiation………………………………………………..………100
4. SDF-1α Upregulated Cells Secrete SDF-1α…………………………102
5. SDF-1 Increases Migratory Capabilities In Vitro……………………102
6. SDF-1α Increases Cell Survival…………………………………..…103
7. SDF-1α and Control Cells Differentiate into Functional Neurons In
Vitro………………………………………………………………….104

8. SDF-1α Increases Differentiation…………….……………..….……105
C. Discussion……………………………………………………………...……107
Chapter VIII. SDF-1α Upregulation in Transplanted iPSC-NPCs Increases
Regeneration and Functional Recovery in an Ischemic Stroke Model
….....………113
A. Introduction……………………………………………………………….…114
B. Results…………………………………………………………………….…116
1. SDF-1α is Upregulated Endogenously After Stroke…………..….…116
2. Exogenous SDF-1α Increases Neurogenesis in the Peri-Infarct
Area…………………………….……………………………………..118
3. iPSC-NPCs With and Without SDF-1α Upregulation Exhibit Neuronal
Differentiation In Vivo……………………..…………………......…119
4. Transplantation of SDF-1α iPSC-NPCs Increased Endogenous
Angiogenesis.…………………………..………………………..…...120
5. Transplantation of iPSCs In Vivo Increases Functional Recovery:
Locomotion......………..………………………..…………………....122
6. Transplantation of iPSCs In Vivo Increases Functional Recovery:
Sensorimotor………...………...………..……………………..……..125
C. Discussion…………………………………………………………………...128
Chapter IX. Summary and Conclusions……………………………………………..134
Chapter X. References………………………………………………………………...137

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