Induced Pluripotent Stem (iPS) Cell Transplantation as a Treatment for Ischemic Stroke Open Access
Chau, Monica Jannie (2014)
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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