selective ablation of transgenic neural precursor cells- a ......pgk: phosphoglycerate kinase...
TRANSCRIPT
Selective Ablation of Transgenic Neural Precursor Cells- A Cellular and Molecular Tool for Use in Neural
Regeneration Studies
by
Christopher Edward Rodgers A thesis submitted in conformity with the requirements
for the degree of Master of Science
Institute of Medical Science University of Toronto
© Copyright by Christopher Edward Rodgers 2018
ii
Selective Ablation of Transgenic Neural Precursor Cells- A
Cellular and Molecular Tool for Use in Neural Regeneration
Studies
Christopher Edward Rodgers
Master of Science
Institute of Medical Science
University of Toronto
2018
Abstract
Induced pluripotent-stem cell derived-neural precursor cell (iPSC-NPC) transplantation
represents a promising potential therapy for repair and recovery of the devastating condition of
spinal cord injury (SCI). Three critical questions on NPC transplantation are (1) "Which neural
cell type provides optimal regeneration?" (2) "Is long term cell integration critically necessary
for recovery or only short term trophic support/immunomodulation?" and (3) “How much
proliferation of transplanted iPSC-NPCs is required for neuroregeneration?” Selective cell
ablation, using enzyme-prodrug systems, such as herpes-simplex virus thymidine kinase (HSV-
TK) and ganciclovir (GCV) or brivudine (BVDU), or nitroreductase (NTR) and metronidazole
(Mtz), is a cellular/molecular tool that may assist in answering the above questions via targeted
ablation of transplanted cells in SCI-model rodents and assessment of repair/recovery. This
project demonstrates successful creation (through transposon gene transfer) and the first
GCV/BVDU-mediated in vitro ablation (>80% reduction over controls) of HSV-TK-expressing
transgenic human iPSC-NPCs in an SCI transplantation context.
iii
Acknowledgments
I wish to express special and particular recognition for my close family members and relatives
who provided constant loving support: my mother, Marilyn Snell; my father, David Rodgers; my
sister, Laura Rodgers; and my grandmothers, Stella Rodgers and Jean Peters.
Additional acknowledgements include, but are not limited to:
My committee members- Dr. Cindi Morshead and Dr. Andras Nagy: For their continued
thoughtful guidance and nurturing support
My supervisor- Dr. Michael Fehlings: For providing the opportunity, environment, and stipend
financial support to conduct my MSc research
My laboratory mentors- Dr. Mahmood Chamankhah, for his exceptional tutelage in conducting
my molecular biology subcloning experiments; and Dr. Mohamad Khazaei for his mentorship in
relation to cell culture techniques.
iv
Table of Contents
Acknowledgments.......................................................................................................................... iii
Table of Contents ........................................................................................................................... iv
List of Abbreviations .................................................................................................................... vii
List of Figures ................................................................................................................................xv
List of Tables .............................................................................................................................. xvii
List of Appendices ..................................................................................................................... xviii
Chapter 1 : Literature Review ..........................................................................................................1
Traumatic Spinal Cord Injury .....................................................................................................1
1.1 Background and Definitions ................................................................................................1
1.1.1 Historical Background and Injury Phases ................................................................1
1.1.2 Temporal Phases of Injury .......................................................................................2
1.2 Mechanisms of Damage in SCI ...........................................................................................3
1.2.1 Demyelination ..........................................................................................................3
1.2.2 Neuronal Loss/Damage ............................................................................................4
Therapeutic Strategies: Neuroprotection and Neuroregeneration ...............................................7
2.1 Cell Transplantation Strategies ..........................................................................................10
2.1.1 Remyelination ........................................................................................................12
2.1.2 Neuronal Regeneration/Axonal-Synaptic Plasticity ..............................................14
Cell Ablation Strategies ............................................................................................................19
3.1 Ablation Studies .................................................................................................................19
3.1.1 Diptheria Toxin Receptor ......................................................................................20
3.1.2 Herpes-Simplex Virus Thymidine Kinase and Ganciclovir ..................................23
3.1.3 Herpes-Simplex Virus Thymidine Kinase and Brivudine .....................................27
3.1.4 E. coli Nitroreductase (NfSA/NfsB) and CB1954 .................................................30
3.1.5 E. coli Nitroreductase (NfsB/NTR) & Metronidazole (Mtz) .................................33
v
3.2 Gene Transfer Vectors .......................................................................................................39
3.2.1 Lentiviral Vectors ..................................................................................................39
3.2.2 Transposon Vectors ...............................................................................................41
Chapter 2: Research Aims .............................................................................................................46
Overarching Goals, Questions, & Technical Aims ........................................................................46
Chapter 3: Materials and Methods .................................................................................................48
1) NTR Insert Subcloning: Lentivirus->PiggyBac ..................................................................48
2) NTR-NPC Monoclonal Lines and Mtz Ablations ...............................................................57
3) NTR-Mtz Ablation Troubleshooting ...................................................................................60
4) HSV-TK Plasmid DNA Purification and Confirmation ......................................................62
5) Mixed NTR and HSV-TK NPC GCV Ablation ..................................................................63
6) HSV-TK-NPC GCV and BVDU Bystander Ablations .......................................................64
7) HSV-TK-NPC GCV and BVDU Quantitative Ablations ....................................................65
Chapter 4: Results ..........................................................................................................................67
1) NTR-PiggyBac Subcloning .................................................................................................67
2) Mtz Treatment of CMV-NTR NPCs ...................................................................................76
3) NTR-Mtz Ablation System Troubleshooting ......................................................................77
4) HSV-TK Plasmids and the Creation of HSV-TK NPCs .....................................................79
5) GCV Bystander Effect: Mixed HSV-TK & HCO-NTR-NPCs ...........................................81
6) GCV/BVDU Bystander Effects: Mixed HSV-TK & WT-NPCs .........................................82
7) GCV and BVDU Successfully Ablate HSV-TK hiPSC-NPCs ...........................................84
Chapter 5: General Discussion, Future Directions & Conclusion .................................................89
5.1 Summary of HSV-TK+GCV/BVDU Results .....................................................................89
5.2 HSV-TK+GCV/BVDU Ablation in Context ......................................................................90
5.3 Advantages and Challenges of HSV-TK+GCV/BVDU Ablation ......................................91
5.4 Reflections on NTR-Mtz Ablation Approaches ..................................................................93
vi
5.5 Potential Applications and Future Directions of HSV-Tk+GCV/BVDU hiPSC-
Derived NPC Ablation .......................................................................................................96
5.6 Conclusion ........................................................................................................................101
References ....................................................................................................................................102
1.1 102
1.1.1 102
1.1.2 104
1.2 104
1.2.1 104
1.2.2 105
2 106
2.1 107
2.1.1 108
2.1.2 110
3.1 114
3.1.1 114
3.1.2 116
3.1.3 121
3.1.4 123
3.1.5 127
3.2 130
3.2.1 130
3.2.2 131
Chapter 3 - 4) ..........................................................................................................................135
Chapter 3 - 7) ..........................................................................................................................136
Appendix I: List of Contributions ................................................................................................137
vii
List of Abbreviations
(In Alphabetical Order)
1O2: singlet oxygen
a.a.: amino acid
AcMNPV: Autographica californica nuclear polyhedrosis virus
adpNSC: adult primitive neural stem cell (GFAP-, Oct-4+)
AIF: apoptosis-inducing factor
ANOVA: analysis of variance (statistical test)
AP: action potential
ASIC: acid-sensing ion channel
Bcl-2: B-cell lymphoma 2
Bcl-xL: B-cell lymphoma-extra large
BDNF: brain-derived neurotrophic factor
BFGF/FGF-2: basic fibroblast growth factor
bp: base pair
BVDU: (E)-5-(2-bromovinyl)-2'-deoxyuridine/brivudine
C11-C14: CMV-NTR-piggybac clones 11-14
CB1954: 5-(aziridin-1-yl)-2,4-dinitrobenzamide (NTR ablation cancer prodrug)
CD95: cluster of differentiation 95
chABC: chondroitinase ABC/chondroitin ABC-lyase
viii
cIAP2/BIRC3: cellular inhibitor of apoptosis protein 2/baculoviral IAP repeat-containing
protein3
CMV-NTR-NPC: ubiquitious promoter-driven-nitroreductase (E. coli NfsB)-expressing neural
precursor cells
CNS: central nervous system
CNTF: ciliary neurotrophic factor
Cre: Causes recombination/Cyclicization recombination
CSPG: Chondroitin-sulphate proteoglycan
CST: cortical spinal tract
Cx43: connexin 43
D5: DCX-NTR-piggyBac clone 5
DAPI: 4′,6-diamidino-2-phenylindole
DCs: dendritic cells
DCX: doublecortin
dCyd: 2'-deoxycytidine
DDH2O: Double-Distilled Water
DISC: death-inducing signalling complex
DMSO: dimethyl sulfoxide
dNSC: definitive neural stem cell (GFAP+, Oct-4-)
dNTP: deoxynucleotide triphosphate
DT: diptheria toxin
ix
dThd: deoxythymidine
DTR: diptheria toxin receptor
dUrd: 2(1)-deoxyuridine
EAA: extracellular amino acid
ECM: extracellular matrix
EDTA: ethylenediaminetetraacetic acid
EGF: epidermal growth factor
EM: electron microscopy
ER: endoplasmic reticulum
ESC: embryonic stem cell
EtOH: Ethanol
FADD: Fas-associated death domain protein
FAF1: Fas-associated factor 1
Fas/FasR: first apoptosis signal/first apoptosis signal receptor
FBS: fetal bovine serum
FMN: flavin mononucleotide
FP: few polyhedral
gag: group antigens lentiviral gene
Gal-1: galectin-1
GCV: ganciclovir = 9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl] guanine
x
GDEPT: Gene-directed enzyme-prodrug therapy
GDNF: glial-derived neurotrophic factor
GFAP: glial fibrillary-acidic protein
GFP: green fluorescent protein
GJIC: gap junction intercellular channel
hat: hobo/Ac/Tam3 transposon family
HB-EGF: heparin-binding EGF-like growth factor
HCO-NTR: human-codon-optimized nitroreductase/NfsB
HCO-NTR-NPCs: Human-codon-optimized-NPCs (expressing NTR under the ubiquitous CMV
promoter)
hCTL: human cytoxic T lymphocyte
HEK-293T: human embryonic kidney 293T cell line
HeLa cells: Henrietta Lacks’ cells
hiPSC: human induced pluripotent stem cell
HIV-1: human immunodeficiency virus 1
hpf: hours post-fertilization
HSV-1: human herpes virus 1
HSV-TK/HSV-TK 1: herpes-simplex virus thymidine kinase 1
•HO: hydroxyl radical
ID2: inhibitor of DNA-binding protein two
ID50/IC50: 50% inhibitory dose/concentration (of viral plaque formation)
xi
iDTR: inducible diptheria toxin receptor
IL-1: interleukin 1
Il-18: interleukin 18
IL-1β: interleukin 1 Beta
IL-6: interleukin 6
INS: preproinsulin gene
IP3: inositol trisphosphate receptor-type 3
iPSC: induced pluripotent stem cell
IR: internal repeat
ITR: inverted terminal repeat
JAK/STAT3: janus kinase/signal transducers and activators of transcription-3
KA: kainite
kb: kilo-base pairs
LB: Lennox-broth
LCs: Langerhans cells
LF: lateral funiculus
LINES: long-interspersed nuclear elements
LTR: long terminal repeat
M1-M5: MB-NTR-piggyBac clones 1-5
MAG: myelin-associated glycoprotein
xii
MAPK10/JNK3: mitogen-activated protein kinase 10/C-Jun-N-terminal kinase 3
MAPK8/JNK1: mitogen-activated protein kinase 8/C-Jun-N-terminal kinase 1
MBP: myelin basic protein
MGE: medial ganglionic eminence
MSC: mesenchymal stem/stromal cell
MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
Mtz: metronidazole
NAD (NAD+/NADH = oxidized vs reduced forms): nicotinamide adenine dinucleotide
NADPH: nicotinamide adenine dinucleotide phosphate
nef: negative factor
NG2: neuron-glial antigen 2
NPC: neural precursor cell (heterogenous mixture of neural progenitor and neural stem cells)
NRP-1: neuropilin-1
NSC: neural stem cell (“upstream” of NPC in differentiation/development)
NTR: Nitroreductase (E. Coli NfsA/NfsB)
NTR-NPC: nitroreductase (E. coli NfsB)-expressing neural precursor cells
OEC: olfactory ensheathing cell
ONOO−: peroxynitrite
OPC: oligodendrocyte progenitor cell
OPI: overproduction inhibition
xiii
P/S: penicillin-streptomycin mixture
p53/TRP53: transformation/tumour-suppressor-related protein 53
PARP1: Poly [ADP-ribose] polymerase 1
PDGF-A: platelet-derived growth factor subunit A
PFA: paraformaldehyde
PFT-μ: pifithrin-μ
PGK: phosphoglycerate kinase
PI3K/Akt/mTOR: phosphoinositide 3-kinase/ Protein kinase B/mechanistic target of rapamycin
pNSC: primitive neural stem cell (GFAP-, Oct-4+)
pol: reverse transcriptase and integrase lentiviral gene
PTEN: Phosphatase and tensin homolog
PTK: puromycin-resistance-HSV-TK fusion protein gene
RCF: relative centrifugal force (often referred to as “g”s)
RCL: replication competent lentivirus
RFP: red fluorescent-protein
RIPA: radioimmunoprecipitation assay
RRE: Rev response element
SAPs: self-assembling peptides
SB: Sleeping Beauty transposon
SCI: spinal cord injury
xiv
SDS-PAGE: sodium-dodecyl-sulfate polyacrylamide gel electrophoresis
Sema3A: semaphorin 3A
SF9: serum free media 9 (components described in Chapter 3: Materials and Methods,
Section 2)
Shh: Sonic Hedgehog
SIN: Self-inactivating
SINES: short-interspersed nuclear elements
1O2: singlet oxygen
•O2-: superoxide
tat: trans-activator lentiviral gene
TE: transposable element
TNF-α: tumor necrosis factor alpha
TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling
vif: viral infectivity
VLF: ventrolateral funiculus
vpr: virus protein R
vpu: virus protein U
VSV-G: vesicular stomatitis Indiana virus G-protein
WT: wild-type
WT-NPC: wild-type hiPSC-derived NPC (no transgene)
xv
List of Figures
Figure C1-I: An Overview of SCI Pathophysiology (pg. 6)
Figure 1a: CMV-NTR Lentiviral Plasmid and Subcloning Insert Gene Maps (pg. 68)
Figure 1b: DCX-NTR Lentiviral Plasmid and Subcloning Insert Gene Maps (pg. 69)
Figure 1c: MBP-NTR Lentiviral Plasmid and Subcloning Insert Gene Maps (pg. 70)
Figure 1d: PiggyBac Transposon Plasmid Vector Gene Map (pg. 71)
Figure 1e: CMV-NTR-PiggyBac Subcloned Plasmid (pg. 72)
Figure 1f: DCX-NTR- PiggyBac Subcloned Plasmid (pg. 73)
Figure 1g: MBP-NTR- PiggyBac Subcloned Plasmid (pg. 74)
Figure 2: NTR Inserts/PiggyBac Transposon Agarose DNA Gel Restriction Digests (pg. 75)
Figure 3: CMV-NTR-NPC Mtz Ablation Attempts (pg. 76)
Figure 4: CMV-NTR-NPC Coomassie Blue Protein Stain (pg. 77)
Figure 5: HCO-NTR-NPC Mtz Ablation Attempts (pg. 78)
Figure 6a: pCMV-Tol2 Plasmid Gene Map (pg. 79)
Figure 6b: pKTol2P-PTK Plasmid Gene Map (pg. 80)
Figure 7: HSV-TK + GCV Bystander Effect with HSV-TK and HCO-NTR NPCs (pg. 81)
Figure 8: HSV-TK + GCV Bystander Effect with HSV-TK and WT-GFP NPCs (pg. 82)
Figure 9: HSV-TK + BVDU Bystander Effect with HSV-TK and WT-GFP NPCs (pg. 83)
Figure 10: Evaluation of GCV ablational efficiency of HSV-TK NPCs (pg. 84)
Figure 11: Evaluation of BVDU ablational efficiency of HSV-TK NPCs (pg. 85)
Figure 12: The Comparison of GCV & BVDU ablational efficiency of HSV-TK NPCs (pg. 86)
xvi
Figure 13: The Evaluation of DMSO treatment of HSV-TK NPCs (pg. 88)
xvii
List of Tables
Table 1: An Overview of key therapeutic neuroprotective and neuroregenerative strategies for
SCI (pg. 9)
Table 2: An Overview of Cell Ablation Systems (pg. 38)
xviii
List of Appendices
Appendix I: List of Contributions: (pg. 137)
1
Chapter 1 : Literature Review
Traumatic Spinal Cord Injury
1.1 Background and Definitions
1.1.1 Historical Background and Injury Phases
The vertebrate spinal cord, or medulla spinalis as it was once referred to, which in humans spans
the cerebrospinal axis from the medulla oblongata to the lowest lumbar vertebra, has long been
known to provide connections between the brain and the peripheral nervous system, including
the motor, sensory, and autonomic nerves as evidenced by forms of paralysis and loss of bladder
function accompanying injury to the cord (Barlow, 1832; Wilson, 1911; Budd, 1839; Ott, 1879;
Nichols, 1899; Pollock et al., 1951; Garstang and Miller-Smith, 2007; Stanley, 1833; Thorburn,
1888). Spinal cord injury (SCI) is defined simply as any damage causing a temporary or
permanent alteration in the cord’s function and can be divided into two principal classes of injury
- namely traumatic injuries encompassing all physical external sources (falls, motor-vehicle
accidents, projectile-wounds, etc.) and non-traumatic injuries encompassing internal disease
sources (vertebral disc degeneration, cancerous tumour, etc.) (reviewed in Ahuja et al.,
2017a&b). With regards to traumatic spinal cord injury, there exist two main phases: primary
and secondary injury (Tator and Fehlings, 1991).
The primary phase of traumatic SCI contains the initial physical damage from such forces as
laceration, shearing, and often most prominently, compression/contusion and is notable
specifically for marking the injury severity (Tator, 1995; Tator and Fehlings, 1991; reviewed in
Ackery et al., 2004). Depending on trauma severity, nerve fiber neuronal axons are either
demyelinated or disintegrated entirely (McDonald, 1975). Subsequently, a secondary phase of
injury, defined as a “delayed and progressive tissue energy” is generated consisting of a cascade
cycle of neuronal and glia cell death as well as inflammation and ischemia (reviewed in Ahuja et
al., 2017a&b). Secondary injury is known to involve both a buildup of extracellular amino acids
(EAAs), particularly glutamate and aspartate that alter neural and glial metabolism, and are
correlated with apoptosis, and inflammation, as well as a disruption of the blood-spinal cord
barrier involving the recruitment of cells of the immune system-including neutrophils,
2
macrophages/microglia and mast cells, which produce pro-inflammatory cytokines, such as
interleukin 6 (IL-6), interleukin 1-Beta (IL-1β), and tumor necrosis factor alpha (TNF-α)
(Hachem et al., 2017; Saghazadeh and Rezaei, 2017; reviewed in Ahuja et al., 2017a&b). The
levels of pro-inflammatory cytokines have been shown to reach a climax between six to twelve
hours post-injury and plateau for as long as an additional four days (Nakamura et al., 2003;
Ulndreaj et al., 2016).
1.1.2 Temporal Phases of Injury
The time length post injury determines the injury phase, of which in current classifications exist
four: acute, sub-acute, intermediate, and chronic, which occur at periods of under forty-eight
hours, forty-eight hours to fourteen days, fourteen days to six months, and beyond six months
respectively (reviewed in Ahuja et al., 2017a&b). During the acute phase, the aforementioned
secondary injury begins to occur as the disruption of the blood-spinal cord barrier from the initial
primary insult produces swelling and hemorrhage as well as vascular disruption and permits the
entry of immune cells and subsequent production of proinflammatory, as well as pro-apoptotic
factors (reviewed in Ahuja and Fehlings, 2016). As a result of the initial inflammation produced
in the acute phase, degradation and death of neurons and oligodendrocytes occur, thus inducing
both demylination and reduced neuronal signaling (reviewed in Ahuja and Fehlings, 2016).
For the twelve days of the subacute phase, arterial spasms, and resultant vasoconstriction, in
addition to vesicular clotting, and fluid-buildup swelling occur, leading to further ischemia,
cutting off vital oxygen and glucose to neurons and glia, further promoting necrosis (reviewed in
Ahuja and Fehlings, 2016; reviewed in Ahuja et al., 2017a&b). The effects of microglia and
other immune cells at the two-week mark post-injury (late subacute-early intermediate phase)
additionally include extracellular damage, leading to the development of cystic microcavities
(reviewed in Ahuja and Fehlings, 2016; reviewed in Ahuja et al., 2017a&b). Further secondary
injury damage and regeneration barriers occur from the result of rapid astrocytic proliferation
proximal to the lesion, as they secrete both proinflammatory factors and extracellular matrix
(ECM) molecules known as chondroitin sulphate proteoglycans (CSPGs) (reviewed in Ahuja
and Fehlings, 2016; reviewed in Fehlings et al., 2017; Rhodes & Fawcett, 2004). The CSPGS
and perilesional astrocytes eventually produce a structure known as the glial scar, which
continues to mature throughout the intermediate and into the chronic SCI phase and presents a
3
critical barrier to neural repair and regeneration (reviewed in Ahuja and Fehlings, 2016;
reviewed in Ahuja et al., 2017a&b). Apoptosis in SCI secondary injury is further promoted due
to an excessive cellular calcium buildup occurring due to loss of ionic homeostasis (Schanne et
al., 1979). These overly high calcium levels activate calcium-dependent proteases, and
negatively impact mitochondria, culminating in programmed cell death (Schanne et al., 1979).
As time progresses to the intermediate phase (two weeks), there occurs a continuation of axonal
death and a coalescence of cystic microcavities to form a larger macrocavity bounded by the
astrocytes and CSPGs of the glial scar, which significantly impedes the regrowth of neuronal
axons, remyelinating projections of oligodendrocytes, and neuronal/glial cell migration to
replace injured or destroyed cells (reviewed in Ahuja and Fehlings, 2016; reviewed in Ahuja et
al., 2017a&b). Despite the hurdles to regeneration created through the maturation of the glial
scar, there is however evidence of some beneficial properties of the scar existing, including the
sealing and protection of the blood-spinal cord barrier, partially-sequestering inflammation, and
preventing further increase of lesion volume (Saghazadeh and Rezaei, 2017).
1.2 Mechanisms of Damage in SCI
1.2.1 Demyelination
The loss and degradation of myelin sheaths coating neuronal axons ranks highly among the most
critical neurological damage conditions induced by SCI (Huang et al., 2014; Wu and Ren, 2009).
Demyelination in SCI is understood to be largely a result of oligodendrocyte apoptosis, which is
believed to be initiated through cellular and biochemical pathways involving the organelles of
the mitochondria and endoplasmic reticulum (ER) of oligodendrocytes (Huang et al., 2014).
Evidence for ER and mitochondrial involvement in oligodendrocyte apoptosis post-SCI can also
be inferred from similar interaction that occur in other forms of central nervous system (CNS)
demyelination, such as that occurring from intracerebral hemorrhage (Zhuo et al., 2016).
In observations of oligodendrocyte death taking place after SCI, evidence of the occurrence of
apoptotic events has been demonstrated from detection of upregulated expression of the proteins
caspase-12, known to promote activation of the inflammatory cytokines interleukin 1 (IL-1) and
interleukin 18 (IL-18), and cytochrome C, known to stimulate apoptosis through binding with
the inositol trisphosphate receptor-type 3 (IP3) receptor in the ER, and inducing the release of
4
stored excess intracellular calcium (Huang et al., 2014; Fisher et al., 2002; Liu et al., 1996). One
major regulator of oligodendrocyte apoptosis has been hypothesized to be the inhibitor of DNA-
binding protein two (ID2), as significant upregulations of ID2 that increase with time post-injury
have been detected in the white matter in rodent models of compressive SCI (Huang et al.,
2014). As ID2 is also thought to be involved in suppressing cellular differentiation, it is possible
that ID2 may also act to prevent oligodendrocyte progenitor cell (OPC) differentiation and
inhibit remyelination during SCI secondary injury (Hara et al., 1994).
In addition to ID2, upregulation of the transformation/tumour-suppressor-related protein 53
(p53/TRP53) appears to play a critical role in oligodendrocyte apoptosis, and thus demyelination
in SCI (Ma et al., 2017). Levels of the transcription factor, E2F1, itself understood to be involved
in both cell cycle control and tumor suppression, have been shown to be upregulated along with
caspase-12 and cytochrome C during oligodendrocyte apoptosis (Ma et al., 2017; Stevens and La
Thangue, 2005). E2F1 expression appears to be partly regulated by p53, as demonstrated by
experiments involving the treatment of SCI-model rats with the p53 chemical inhibitor, pifithrin-
μ(PFT-μ), which resulted in both genetic/protein-based changes: the suppression of caspase-12,
cytochrome C and E2F1, along with physiological improvements: the increase in prevalence,
thickness, and length of myelin sheaths of spared axons (Ma et al., 2017). Demyelination clearly
represents a formidable, yet hopefully not insurmountable challenge along the road to repair and
recovery in traumatic SCI.
1.2.2 Neuronal Loss/Damage
The damage to and loss of neurons following traumatic SCI is an issue of serious pathological
consideration (reviewed in Ahuja et al., 2017a&b). The marked increase of extracellular
glutamate is known to be one of the factors of neuronal excitotoxicity and resultant apoptosis
(Mazzone et al., 2017; reviewed in Ahuja et al., 2017a&b). Glutamate excitotoxicity in spinal
neurons has been shown to be a slow and gradual process, with a correlation between increasing
cell loss of grey matter motor and premotor spinal neurons with increasing concentrations of the
administered glutamate analog kainite (KA; between 0.01mM-0.05mM and beyond) (Mazzone
et al., 2010).
Recent work has provided insight into the mechanism of extracellular glutamate-related neuronal
loss/damage, showing that the glutamate surge may activate acid-sensing ion channels (ASICs)
5
on SCI neurons, through an extracellular pH reduction (Mazzone et al., 2017). Treatment of
mouse spinal cord slices with KA at a concentration of 0.01mM, right at the threshold for
excitotoxic damage, has been demonstrated to increase gene expression of the ASICs: ASIC1a,
ASIC1b, ASIC2 and ASIC3, whereas a treatment with a ten-fold increase in KA concentration
actually reduced gene expression of the ASICs: ASIC1a and ASIC2 (Mazzone et al., 2017).
Taken together, the aforementioned results would appear to indicate that the excessive buildup of
extracellular glutamate in traumatic SCI secondary injury may facilitate a reduction in the
excitoxicity threshold of spinal neurons, through downregulation of ASIC expression, and thus
make them more susceptible to undergo excitotoxic apoptosis in the presence of high levels
(>0.01mM) of extracellular glutamate (Mazzone et al., 2017).
Oxidative stress presents an additional mechanism behind the neuronal loss exhibited in cases of
traumatic SCI, as it does in other forms of CNS damage/degeneration (reviewed in Smith et al.,
2013). Reactive oxygen species, including such molecules as hydroxyl radicals (•HO),
peroxynitrite (ONOO−), superoxide (•O2-), and singlet oxygen (1O2), which are naturally
produced during cellular metabolism of oxygen, rise in concentration as part of the secondary
injury cascade of traumatic SCI (as similarly occurs in other forms of CNS traumatic injury), and
induce cellular damage/death of spinal neurons through such mechanisms as lipid peroxidation
of the cell membrane, DNA damage, and protein oxidation (reviewed in Smith et al., 2013;
reviewed in Catala, 2009; Ansari et al., 2008).
Necrosis, or delayed cell death of neurons is generally more prevalent than apoptosis as a result
of oxidative stress in traumatic SCI, and acts in a “spreading” manner leading to necrosis in
neighbouring neurons (Muthaiah et al., 2017). In necrosis, it is understood that the DNA binding
protein, Poly [ADP-ribose] polymerase 1 (PARP1) is upregulated, and recent research has
discovered that the cellular enzymes mitogen-activated protein kinase 8/C-Jun-N-terminal kinase
1 (MAPK8/JNK1) and mitogen-activated protein kinase 10/C-Jun-N-terminal kinase 3
(MAPK10/JNK3) are involved in PARP1-induced neuronal necrosis (Muthaiah et al., 2017).
It was previously reported that Fas-associated factor 1 (FAF1) stimulates PARP1 expression, via
reactive oxygen species interaction with FAF1 resulting in its transport to the nucleus and
catalytic induction of PARP1 gene expression (Yu et al., 2016). In addition to its presence
promoting PARP1 expression and necrosis, FAF1 depletion was observed to prevent the
6
downstream PARP1-mediated necrotic event of energetic collapse (e.g., mitochondrial
depolarization and nuclear translocation of apoptosis-inducing factor (AIF)) (Yu et al., 2016). In
order to provide neural repair for traumatic SCI, and the resultant neuronal damage and loss from
secondary injury, mitigating or reducing necrosis due to oxidative stress would appear to be a
potentially beneficial, and efficient strategy.
Figure C1-I: An Overview of SCI Pathophysiology. SCI consists of an initial physical trauma
(primary injury, shown in a) on the right-middle), followed by an extended damaging process of
secondary injury that is subdivided into 4 temporal phases post-injury (a-c). The acute phase (a): 0-
48hrs) involves vesicular damage and blood-spinal cord barrier disruption resulting in hemorrhage,
ischemia, edema, as well as the infiltration of immune cells (e.g., neutrophils, monocytes, etc.), and
apoptotic/necrotic neuronal and glial cell death (leading to demyelination and axonal
severing/degeneration). During the subacute phase (b): 48hrs-14 days), demyelination, axonal
degeneration, and neuroinflammation (e.g., macrophage maturation from monocytes) continue and
the processes of cavity formation and reactive astrogliosis begin with the first deposition of CSPGs.
Throughout the intermediate phase (c): 14 days- 6 months) microcavities grow and coalesce as the
lesion matures to form a central macro-cavity bounded by a glial scar composed of astrocytes and
CSPGs. By the chronic phase (c): 6 months and beyond), glial scar maturation and lesion
stabilization occur, in addition to cyst formation due to clearance of microglial debris, and the gradual
removal of damaged neuronal axons and soma.
Image reproduced with permission from: Ahuja et al., 2017b. Neurosurgery. PMID: 28350947
C1-I
a) b) c)
7
Therapeutic Strategies: Neuroprotection and Neuroregeneration
Two major strategies towards pursuing treatment for traumatic SCI are neuroprotection, defined
as “the attenuation of pathophysiological processes triggered by acute injury to minimize
secondary damage,” and neuroregeneration, which alternatively seeks to repair and replace
damaged neural tissue and provide functional recovery (reviewed in Hilton et al., 2017; reviewed
in Ahuja and Fehlings, 2016). The three major neuroprotective treatments currently in use
clinically are: surgical decompression, methylprednisolone (or other neuroprotective agents)
administration, and blood pressure augmentation (reviewed in Ahuja et al., 2017). There are
additionally several experimental treatments currently being explored (reviewed in Ahuja et al.,
2017b; reviewed in Hilton et al., 2017).
Although the strategy of neuroprotection has and likely will continue to provide strong
therapeutic benefit for reducing secondary injury damage in traumatic SCI, its main drawback
and limitation lies in the simple fact that it can only preserve neural tissue, and not repair or
restore damaged or lost tissue, nor provide full functional recovery (reviewed in Hilton et al.,
2017; reviewed in Ahuja et al., 2017b; reviewed in Ahuja and Fehlings, 2016; reviewed in Ahuja
et al., 2016). While neuroprotection aims to mitigate the neuronal and glial damage contributing
to sensory and motor loss in traumatic SCI, neuroregeneration proposes to replace damaged or
dead neurons and oligodendrocytes and/or provide restored neuronal connectivity and synaptic
activity through outgrowth, rewiring, and remyelination of existing axons (reviewed in Hilton et
al., 2017; reviewed in Ahuja et al., 2017b; reviewed in Ahuja and Fehlings, 2016; reviewed in
Ahuja et al., 2016; reviewed in O’Donovan, 2016).
Three critical neuroregenerative strategies for traumatic SCI being investigated in animal models
include (1) remyelination of surviving neuronal axons, (2) neuronal plasticity enhancement
through neuronal replacement and/or rewiring of existing axons and synaptic connections, and
(3) glial scar degradation/modification (reviewed in Ahuja et al., 2017b; reviewed in Ahuja and
Fehlings, 2016; reviewed in Ahuja et al., 2016).
8
Cell replacement for neurorepair can involve either endogenous activation (mobilization,
migration/transportation, and differentiation) of resident neural stem/progenitor (the mixture
hereafter referred to as neural precursor cells (NPCs)) cells, or transplantation of exogenous
NPCs or OPCs (for purely remyelination based approaches) (reviewed in Ahuja et al., 2016;
reviewed in Franklin, 2015; Najm et al., 2015). With regards to exogenous NPC/OPC
transplantation, it is additionally not currently understood whether short-term trophic support
from transplanted-cell-secreted factors/molecules/proteins etc., or long-term integration of
transplanted cells represent the more critical mechanism of regeneration, and thus the more
efficient approach for neurorepair (reviewed in Ahuja et al., 2017b; reviewed in Ahuja and
Fehlings, 2016; reviewed in Ahuja et al., 2016). The precise amount and required time length of
transplanted cell (e.g., NPC, OPC, etc.) proliferation (e.g., how long must proliferation
occur/what cell quantity is necessary for providing physical repair/functional recovery?) is
similarly not entirely understood.
9
A) Neuroprotection B) Neuroregeneration
Pharmacological treatment:
-methylprednisolone (currently used)
-riluzole (in clinical trial)
-minocycline (in clinical trial)
-glyburide (in clinical trial)
Endogenous cell (NPC/OPC) activation:
-Olig2 upregulation
-galectin-1 administration
-cyclic AMP
-rho-associated protein kinase inhibition
-Nogo-A inhibition
Surgical decompression (currently used)
Exogenous NPC-based cell transplantation-
remyelination and/or axonal regeneration:
-NPCs
-OPCs
-neuronal precursors
Moderate hypothermia: -reducing systemic body
temperature to 330C by intravascular cooling
strategies (in clinical trial)
Exogenous cell transplantation- additional cell types:
-olfactory-ensheathing cells
-Schwann cells
-mesenchymal stromal cells
Blood pressure-modulation
(currently used)
Glial Scar modification-
-chABC treatment
-neuron-glial antigen 2 knockdown
Table 1: An Overview of key therapeutic neuroprotective and neuroregenerative
strategies for SCI. While many neuroprotective options (A) are currently employed clinically
or are undergoing/have undergone clinical trials and produce modest sensorimotor benefit,
they are incapable of providing full physical repair and functional recovery. A plethora of pre-
clinical neuroregenerative strategies (B) are currently under investigation in animal models
with widely varying, yet greatly promising results.
10
2.1 Cell Transplantation Strategies
Cell transplantation therapies for traumatic SCI represent a promising option in order to promote
neuronal/axonal regeneration and improved plasticity and synapse formation (reviewed in Nori
et al., 2017). Two critical goals in cell transplantation-based strategies for SCI repair include: (1)
remyelination of injured axons; and, (2) axonal/neuronal regeneration and synaptic
restoration/rewiring (reviewed in Nori et al., 2017; reviewed in Ahuja et al., 2017b).
The microenvironment niche of the injured spinal cord is understood to be a major obstacle for
cell-transplantation based-neuronal regeneration, both in terms of limiting exogenous cell
survival as well as reducing the differentiation of NPCs towards a neuronal lineage, making
axonal regeneration a more difficult task than remyelination with cell transplantation (reviewed
in Nori et al., 2017; reviewed in Ruff et al., 2012). The combination of cell transplantation (e.g.,
NPCs, OPCs, mesenchymal stromal cells, olfactory ensheathing cells, etc.) with
immunomodulation and trophic support, to ameliorate the injured cord microenvironment’s
permissiveness to cell integration and survival as well as promote remyelination, axonal
regeneration, synapse formation, collateral sprouting, and neurogenesis as well as lessen the
degradation of retrograde axons has been proposed and is currently under evaluation in animal
SCI models (reviewed in Ruff et al., 2012; reviewed in Liu et al., 2012; reviewed in Sun and He,
2010; reviewed in McKerracher, 2001).
An elusive major question, in addition to that of the debate of remyelination vs neuronal/axonal
regeneration and synaptic rewiring, that has remained unsolved as of yet, in regards to cell
transplantation-based therapies for promoting remyelination, axonal regeneration and plasticity
for traumatic SCI, is namely, which of the two mechanisms, (1) microenvironment modification
(e.g., immunomodulation) and trophic support, or (2) long-term survival, migration,
differentiation (in the case of NPCs) and integration of transplanted cells, represents the most
critical mechanism that can provide physiological regeneration/repair and functional recovery
(reviewed in Badner et al., 2017; reviewed in Iyer et al., 2017; reviewed in Ruff et al., 2012;
reviewed in Kim et al., 2007).
11
The mechanisms of long-term transplanted cell survival and short-term support (trophic support,
immunomodulation, etc.) are heavily intertwined, wherein current studies cannot usually
separate either as being the primary mediator of reported physical repair (remyelination, axonal
regeneration, reduced astrogliosis, etc) and functional recovery (hindlimb motor performance,
BBB scores etc.) and, as such it is difficult to determine which of the two may provide the most
effective and efficient direction for therapeutic benefit and eventual translation (reviewed in
Bonner and Steward, 2015; reviewed in Lu et al., 2014; reviewed in Li and Lepski, 2013;
reviewed in Ruff et al., 2012; reviewed in Fehlings and Vawda, 2011).
Evidence specifically focusing on the importance of the long-term survival and integration (e.g.,
synaptogenesis etc.) for transplanted stem cells in mediating physical repair and functional
recovery in SCI animal models is generally scarce, but still has been demonstrated through such
experiments as those that displayed the survival of a majority of adult-human spinal-cord derived
neurospheres up to 6 weeks after transplantation in rats, correlating with behavioural
improvements (Akesson et al., 2007). An additional study reported survival of up to 8 weeks of
human autologous, reprogrammed, iPSC-derived NPCs transplanted into thoracic-injured mice,
as well as both their successful differentiation into neurons and glia, and functional integration
(synaptic-connections with surviving endogenous axons and remyelination), all without the
additional use of extra neurotrophic factors, biomaterials-scaffolding, or immune modulation
(Liu et al., 2017).
Additionally, for transplanted proliferating cells, such as human iPSC-derived NPCs, it is
uncertain precisely how much cell proliferation is necessary and for how long it must occur in
order to provide adequate and sufficient physical repair and functional recovery. Previous
research using a rat model of middle cerebral artery occlusion stroke has indicated that
transplanted human iPSC-derived NPCs continue to proliferate (as evidenced by nestin
expression) up to at least 2 weeks post transplantation and can improve neurobehavioural
outcomes, however it is unknown if shorter-term proliferation may be sufficient for providing
adequate neuroregeneration through trophic support and immunomodulation in CNS conditions
(Yuan et al., 2013).
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2.1.1 Remyelination
In order to reduce demyelination and promote remyelination after traumatic SCI, it may be
worthwhile to develop methods to replace dead oligodendrocytes through NPC or OPC
transplantation, as well as reduce/inhibit oligodendrocyte apoptosis, and promote endogenous
OPC differentiation.
In terms of promoting endogenous remyelination, the b-HLH transcription factor and prominent
oligodendrocyte marker, Olig2, known to be significant in mammalian CNS development, has
been identified as a strong candidate for promoting endogenous remyelination (Tan et al., 2017).
Via the use of lentiviral vector gene therapy, Olig2 overexpression was recently shown in a
rodent SCI model to increase differentiation of OPCs to mature oligodendrocytes, induce
remyelination, reverse gene expression changes of several transcription factors whose expression
is altered in secondary injury, and even provide significant improvement in hind limb motor
performance (Tan et al., 2017).
With regards to cell transplantation approaches for promoting remyelination, NPCs of either
adult (endogenous or induced pluripotent) or embryonic origin, present promising transplantation
cell types for neuroregeneration (reviewed in Ahuja et al., 2017b; reviewed in Ahuja and
Fehlings, 2016; reviewed in Ahuja et al., 2016; reviewed in Lu, 2017; reviewed in Lopez Juarez
et al., 2016; reviewed in Doulames, 2016). One previous study from over a decade ago
determined that NPC transplantations in combination with Sonic hedgehog (Shh) were capable
of promoting white matter-tract sparing and functional motor improvements (BBB scores and
motor evoked potentials) in thoracic (T9-T10) contusion injured rats (Bambakidis and Miller,
2004). The work of Bambakidis and Miller particularly found that the combinatorial treatment
provided improved survival, proliferation, and migration of transplanted NPCs.
In order to determine the most effective timepoint for cell transplantation, one set of rodent
model SCI experiments compared the transplantation of OPCs (pre-differentiated from human
NPCs) at either 7 days or 10 months, both of which permitted survival, migration, and
differentiation of OPCs; however only in the 1 week-post injury transplantation animals was
enhanced remyelination or improved motor performance observed (Keirstead et al., 2005).
Another study from the mid 2000’s compared transplantations of NPCs at 2 weeks (subacute
13
phase) and 8 weeks (chronic phase) post-T7 23-gram clip compression injury (Karimi-
Abdolrezaee et al., 2006).
While the above study found that 2 week-post injury transplanted NPCs showed strong survival,
proliferation, migration (>5mm rostrally and caudally of injection point), and differentiation-
prominently into OPCs and subsequently myelinating and myelin basic protein (MBP)-
expressing oligodendrocytes, 10 week-post injury injected NPCs faired relatively poorly in terms
of survival in comparison (Karimi-Abdolrezaee et al., 2006). In addition to NPC survival,
proliferation, migration, and differentiation, Karimi-Abdolrezaee et al. demonstrated axon
ensheathment and even improved functional recovery as detected by improved BBB scores, and
other behavioural grading systems (grid walk) for rats receiving 2-week post-SCI NPC
transplantions compared to vehicle controls.
Subsequent experiments with the same model showed that transplanted-NPC-derived-
oligodendrocytes could within 6 weeks, additionally provide such functional morphological
benefits as forming compact myelin and Nodes of Ranvier (detected via electron micrscopy
(EM)), primarily along white matter tracts and improving conduction speed of action potentials
(APs) on ensheathed axons (observed through electrophysiological probes) (Eftekharpour et al.,
2007). The additional combination of NPC transplantations with epidermal growth factor
(EGF), basic fibroblast growth factor (BFGF), Platelet-derived growth factor subunit A (PDGF-
A) and chondroitinase ABC (ChABC) was later shown to strongly improve survival and
migration of transplanted NPC-derived OPCs and oligodendrocytes (Karimi-Abdolrezaee et al.,
2010). By lentivirally-transducing NSCs with the gene, Olig2, a critical regulator of
oligodendrocyte development, after 7 weeks transplantation, one group was able to show
significantly improved migration towards the white matter, white matter sparing, lesion cavity
volume reduction, OPC proliferation, myelin sheath thickness, and hindlimb motor functional
recovery compared to control rats receiving either NPCs alone or pure vehicle controls (Hwang
et al., 2009).
Ciliary neurotrophic factor (CNTF) has been additionally used as a successful combinatorial
remyelination treatment in rodent constusive SCI models along with OPC transplantation (Cao et
al., 2010). In a study using transgenic-CNTF-expressing OPC transplantations, the
combinatorial treatment was shown to greatly improve: a) OPC survival, proliferation, and
14
differentiation into oligodendrocytes; b) remyelination of axons at the lesion area near the
ventrolateral funiculus (VLF) or lateral funiculus (LF); c) motor-evoked potential recovery; and
d) hindlimb motor performance scores (Cao et al., 2010).
Two more recent studies that made use of either induced-pluripotent stem cell (iPSC)-derived
OPCs, or iPSC-derived NPCs for intraspinal cell transplantation post-contusive thoracic (T10)
SCI in rat models reported major improvements in: a) myelin formation and axon ensheathment;
b) electrophysiological improvements in axon conduction; and c) motor performance (BBB
scores etc.) when compared to vehicle control animals (Salewski et al., 2015; Kawabata et al.,
2016).
Critical among the results of Salewski et al. and Kawabata et al. was the demonstration of some
of the earliest evidence that iPSC-derived NPCs and OPCs can similarly provide successful and
efficient neuroregeneration through remyelination as well as functional motor recovery, just as
previous work proved was possible for NPCs and OPCs of embryonic stem cell (ESC) or
endogenous adult origins (reviewed in Khazaei, et al., 2017; Salewski et al., 2015; Kawabata et
al., 2016; reviewed in Lee-Kubli and Lu, 2015; reviewed in Khazaei et al., 2014). As powerful a
strategy for traumatic SCI neuroregeneration/neurorepair as remyelination may present itself to
be, it may still not be able to provide complete functional recovery, as it does not address the
issue of dead or damaged neuronal axons and destroyed synapses surrounding the lesion area
(reviewed in Ahuja et al., 2017a&b; reviewed in Ahuja et al., 2016). The restoration of full
motor, sensory and autonomic recovery may also require supporting neuronal plasticity- the
regrowth of axons/neurites and re-establishment of synaptic connections.
2.1.2 Neuronal Regeneration/Axonal-Synaptic Plasticity
The restoration of full motor, sensory and autonomic recovery for traumatic SCI may, in
addition to remyelination, require supporting neuronal plasticity- the regrowth or replacement of
dead/damaged neurons, the outgrowth of axons and re-establishment of synapses affected by the
injury (reviewed in Nori et al., 2017; reviewed in Ahuja et al., 2017a&b; reviewed in Ahuja and
Fehlings, 2016; reviewed in Ahuja et al., 2016). In order to accomplish such neuronal plasticity
recovery, a wide variety of strategies, from endogenous activation with administration of growth
factors/hormones, regulatory proteins, or stimulatory/inhibitory molecules, to cell transplantation
(NPCs, Schwann cells, mesenchymal stem/stromal cells (MSCs), olfactory ensheathing cells
15
(OECs), etc.) with methods taken to promote neurogenesis, axonal regeneration, and both
trophic support, and the survival and integration of transplanted cells have been pursued
(reviewed in Nori et al., 2017; reviewed in Ruff et al., 2012; reviewed in Hu and Selzer 2017;
reviewed in Kwon et al., 2002).
Natural, endogenous neuronal regeneration after traumatic SCI is minimal and only occurs for
neurons farther away from the lesion, as has been recently modeled in rats to only occur as close
as 3-5mm away from the SCI lesion area, and thus methods to stimulate endogenous repair, or
transplant cells to replace neurons may be necessary to establish functional recovery (He and
Nan, 2016; reviewed in Ahuja et al., 2017b; reviewed in Bähr and Bonhoeffer, 1994). As myelin
debris, particularly in the form of myelin-associated glycoprotein (MAG) is known to induce
inhibitory effects upon neuronal regeneration, it may be particularly important to examine the
interactions occurring between treatments for neuronal regeneration and remyelination, as there
is some evidence that the latter may potentially alter the former, even possibly detrimentally
(reviewed in Rao and Pearse, 2016).
Various animal SCI model experiments have shown promise in terms of promoting axonal
regeneration and plasticity with transplanted cell types including MSCs, Schwann cells, and
NPCs (of ESC, iPSC, or adult endogenous origin), however of these cell types, only NPCs
possess the capacity to differentiate into all three main neural cell types (neurons,
oligodendrocytes, and astrocytes) and thus they will subsequently represent the primary focus for
this review section (reviewed in Nori et al., 2017; reviewed in Ruff et al., 2012; reviewed in Kim
et al., 2007; Yang et al., 2017; Liu et al., 2013; Ban et al., 2011; reviewed in Oliveri et al., 2014).
Despite the lack of full endogenous regeneration and functional recovery in human (and
mammalian in general) SCI, there is evidence that some endogenous repair does occur, both in
the form of reorganizing surviving axonal connections and synapses and the minor proliferation
of endogenous NPCs; thus one potential focus for cell transplantation based strategies for
neuronal/axonal regeneration and plasticity is to capitalize on and support these endogenous
mechanisms, through trophic support, scaffolding and microenvironment/immune modulation
(reviewed in Bareyre, 2008).
Research involving transgenic NPCs expressing a modified form of the rabies virus (TVA and
G-protein) for synaptic tracing provides perhaps one of the most comprehensive views of the
16
possibilities of long-term synaptic integration of grafted NPC-derived neurons, as there is
evidence of synaptic input from not only corticospinal and reticulospinal neurons of the host
animal, but also from neurons of the cortex, dorsal root ganglion, and medulla oblongata (Adler
et al., 2017). Recent work using mouse ESC-derived NPC grafts to partially regenerate cortical
spinal tract (CST) neurons/axons and recreate functional excitatory synapses after C4 CST
lesions further support the key value of long-term NPC-transplant integration; however the
translational relevance of the study involved may be considered questionable, due to not using a
contusion or compression SCI model, and thus not completely modelling the inhibitory
microenvironment produced by compressive/contusive SCI (Kadoya et al., 2016; reviewed in
Ahuja et al., 2017a&b).
The specific benefits of survival and long-term integration of transplanted NPCs and derived
cells (addressed in Section 2.1 above) have perhaps been highlighted in greater depth in other
CNS-injury models, including recent mouse neuropathic pain model experiments that showed
survival and integration of large numbers of ESC-derived, medial ganglionic eminence (MGE)-
like interneuron precursors up to 6 months after transplantation into the T10-T11 spinal cord,
which not only differentiated largely in to GABA-ergic interneurons and received synaptic inputs
from endogenous axons (primary afferent and spinal cord neurons), but also promoted improved
bladder function, and reduced pain hypersensitivity (Fandel et al., 2016; Etlin et al., 2016;
Hwang et al., 2016; Bráz et al., 2012).
Although such neuropathic pain studies provide valuable insight into the regenerative benefits of
long-term integration of neuronal precursors into the injured spinal cord, stressing the significant
value of focusing efforts on the integration sub-strategy for CNS damage, they do not model
contusive SCI, particularly with its inhibitory inflamed microenvironment, and thus do not
indicate whether integration for other neuronal subtypes (e.g., motor neurons as opposed to
inhibitory interneurons) may be capable of providing similar reparative benefits and functional
recovery (e.g., hindlimb motor function) (reviewed in Ahuja et al., 2017a&b; Fandel et al.,
2016).
The significance of neurotrophic factors in supporting axonal regeneration in addition to other
forms of physical repair is evident from a marmoset SCI model study involving the
transplantation of transgenic NPCs expressing galectin-1 (Gal-1), which not only improved the
17
survival and neuronal differentiation of transplanted NPCs, but also resulted in higher BBB
motor scores (Yamane et al., 2010). The mechanism for Gal-1 mediated axonal regeneration
was determined to occur through Gal-1’s selective binding to the neuropilin-1 (NRP-
1)/PlexinA4 receptor complex, blocking the interaction between semaphorin 3A (Sema3A) and
the NRP-1 receptor complex, and preventing downstream inhibitory signaling-primarily in the
form of excess hydrogen peroxide production and destabilization of F-actin (Quintá et al., 2014;
Quintá et al., 2016).
In a similar vein, there is compelling evidence that microenvironment modifications, such as: (1)
STAT-3 overexpression; (2) inhibition/suppression of the axon guidance molecules and
developmental attractants and repellants of ascending and descending axons respectively, Wnts
1, 4, and 5a (usually absent and known to be reintroduced in the injured cord milieu); (3)
phosphate and tensin homolog (PTEN) inhibition (demonstrated to promote CST axonal axonal
sprouting); (4) IL-6 activation (to stimulate janus kinase/signal transducers and activators of
transcription-3 (JAK/STAT3) and phosphoinositide 3-kinase/protein kinase B/mechanistic
target of rapamycin (PI3K/Akt/mTor); as well as (5) the use of biomaterial-based scaffolds,
including self-assembling peptides (SAPs) to support axonal regrowth may separately be
effective strategies for inducing axonal regeneration and improved plasticity after SCI (Mehta et
al., 2016; Luo et al., 2016; Leibinger et al., 2013a; Leibinger et al., 2013b; Liu et al., 2008; Du et
al., 2015; Liu et al., 2010; Zweckberger et al., 2016; Zweckberger et al., 2015).
Both the directions of (1) long-term survival, differentiation, and integration of transplanted
NPCs/NSCs, and (2) trophic support/scaffolding/immune modulation have shown strong
potential for promoting neuronal regeneration and plasticity in animal SCI models; however
further study may be necessary to separate and compare the differential benefits of each strategy
(reviewed in Nori et al., 2017; reviewed in Ruff et al., 2012; Badner et al., 2017; reviewed in Iyer
et al., 2017). A combination of both strategies, such as shown by studies combining the
transplantation of neuronal and glial-restricted progenitors (a stage between NPCs and mature
neurons/glia) to reconstitute excitatory and inhibitory neurons as well as oligodendrocytes, with
lentiviral-vector administration of brain-derived neurotrophic factor (BDNF) to guide axonal
regeneration in the desired direction (rostral or caudal), will likely be necessary in order to fully
re-establish axonal/neuronal regeneration and synaptic connections and functional motor and
18
sensory recovery (reviewed in Nori et al., 2017; reviewed in Ruff et al., 2012; Bonner et al.,
2011; Bonner et al., 2010).
Furthermore, the issue of the timing and amount of transplanted NPC proliferation required in
order to provide effective physical repair (e.g., remyelination of axons, axonal regeneration or
neuronal replacement and rewiring of synapses, etc.) and functional sensorimotor recovery is
additionally uncertain. Although previous work in rodent CNS injury/condition (e.g., spinal cord
injury and stroke) models has shown functional behavioural improvements using transplanted
iPSC- or ESC-derived NPCs that continued to proliferate up to at least the 2-week post-
transplanation timepoint, what remains unclear is whether such neurological improvements
occurred largely due to trophic and immunomodulatory factor secretion and thus activation of
endogenous regeneration processes (e.g., central canal NPC proliferation, migration, and
differentiation), or actual cell replacement with the integration of the transplanted NPC-derived
neurons and glia themselves (Yuan et al., 2013; Salewski et al., 2013; Karimi-Abdolrezaee et al.,
2010; Kawabata et al., 2016; reviewed in Lee-Kubli and Lu, 2015).
19
Cell Ablation Strategies
3.1 Ablation Studies
The concept of cell ablation represents a potential method for pursuing a better understanding of
the mechanisms of regeneration, as well as developmental roles of specific cells and their lineage
relationships over the course of development (reviewed in Curado et al., 2008). Pertaining to
SCI neurorepair/regeneration, cell ablation studies may be particularly attractive for use in
combination with cell transplantation, for the purpose of providing a method to conduct loss of
function experiments via selectively ablating grafted cells and comparing physical repair and
functional recovery with control animals not receiving ablations. As mentioned previously in
Section 2.1.2, three unanswered questions with regards to neural regeneration are:
1) “Is a) remyelination, or b) neuronal regeneration (neurite outgrowth)/axonal and synaptic
plasticity reestablishment the more effective and efficient strategy for SCI neurorepair (and
eventually pursuing translational therapeutic treatments)?”
2) “Is long-term integration of transplanted cells (e.g., establishment of synaptic connectivity
from transplanted NPC-derived neurons, or large-scale remyelination directly by transplanted
NPC/OPC-derived oligodendrocytes) necessary for providing significant physical repair and
functional recovery, or is the combination of the short-term mechanisms of: a) trophic support
(e.g., secretion of positive growth factors – BDNF/ glial-derived neurotrophic factor (GDNF)
etc.), and b) immune modulation (e.g., downregulating production of Il-6, IL-β, or TNF-α)
sufficient?”
and 3) “How much proliferation of transplanted hiPSC-NPCs is necessary and for how long must
it occur to provide effective SCI physical repair and functional recovery?” (reviewed in Ahuja et
al., 2017b; reviewed in Ahuja and Fehlings, 2016; reviewed in Siddiqui et al., 2015; reviewed in
Badner et al., 2017; reviewed in Khazaei et al., 2017).
20
Studies centered around selective cell ablation of specific types of transplanted cells (e.g.,
neurons and oligodendrocytes), such as those making use of enzyme-prodrug combinations may
provide one avenue for answering, or at least partially clearing up the above questions. Possible
systems for cell ablation include: a) transgenic expression of the diptheria toxin receptor in
combination with diptheria toxin treatment, b) the herpes-simplex virus thymidine kinase-
ganciclovir/brivudine combination, and c) the E. coli nitroreductase (NfsB or NfsA genes)-
metronidazole or CB1954 combinations (reviewed in Curado et al., 2008).
3.1.1 Diptheria Toxin Receptor
Diptheria toxin (DT) is a 535-amino acid dipeptide exotoxin encoded by a bacteriophage
that infects Corynebacterium diphtheriae (Wagner et al., 2002; Pappenheimer 1977; Freeman,
1951). DT’s crystal structure has been determined to be that of a Y-shaped molecule, containing
three domains, that splits into two fragments: (1) fragment A possessing the functional catalytic
(C) domain that transfers an ADP ribose from nicotinamide adenine dinucleotide (NAD) to a
diphthamide residue of eukaryotic elongation factor 2 (eEF-2), thus blocking translation in
infected cells- resulting in the molecule’s toxicity in eukaryotes expressing the receptor; and (2)
fragment B possessing an endosomal translocation (T/TM) domain and a receptor binding (R)
domain on its carboxy-terminal (Bell and Eisenberg, 1997). The cell surface receptor for
diptheria toxin: heparin-binding EGF-like growth factor (HB-EGF), is expressed in humans, but
not in mice, thus rendering the exotoxin largely ineffective against endogenous mouse cells, as it
cannot normally be endocytosed (Saito et al., 2001; Cha et al. 2003). Through transgenic
expression of HB-EGF, the diphtheria toxin cell-surface receptor (DTR) in mouse cells, a
selective/cell-specific ablation system can be set up, whereby DTR-expressing mouse cells (from
embryonic genetic modification or transplantation) or transplanted human cells (naturally DTR-
expressing) can be selectively ablated with DT treatment of experimental mice (Saito et al.,
2001).
The DTR ablation system was first termed a "toxin receptor-mediated cell knockout” by Saito et
al., who used a hepatocyte-specific promoter to induce fulminant hepatitis in transgenic,
hepatocyte-DTR expressing mice (Saito et al., 2001). Saito et al. were able to display a
correlation between hepatocyte sensitivity to DT and transgenic DTR/HB-EGF expression, using
as many as three cell lines (Saito et al., 2001). An inducible variant of transgenic DTR
21
expression (iDTR) was later developed using the Causes recombination/Cyclicization
recombination (Cre)-Lox recombination system, in which mice containing the HB-EGF gene
under the control of a ubiquitous promoter containing a transcriptional stop cassette, were
crossed with a cell-specific (CD4 T cell and CD19 B cell) Cre-expressing mouse line, thus
enabling cell specific removal of the stop cassette and cell-specific DTR/HB-EGF expression
(Buch et al., 2005).
For CD4 T-cell-DTR-expressing mice, Buch et al. demonstrated that 100ng daily injections of
DT over the course of 3 days was sufficient to reduce the mean splenic T:B cell ratio from 0.5 to
0.1 (thus ablating approximately 80% of splenic CD4-T cells); similarly for CD19-B cells, they
displayed that daily doses of 100ng DT reduced the splenic B:T cell ratio from 2.1 to 0.6 after 3
days, and 0.3 after 7 days, and finally 100ng DT doses provided every 8 hours for 7 days resulted
in near 100% ablation of CD19-B cells (Buch et al., 2005).
In regards to neural cell type ablation, Buch et al. not only reconfirmed that DT is capable of
crossing the BBB in mice, but also generated an oligodendrocyte-specific DTR-expressing
mouse line by crossing the ubiquitous-promoter DTR line with a MOG promoter-CRE mouse
line (Buch et al., 2005; Wrobel et al., 1990). Treatment of the MOG-iDTR mice with 100ng of
DT (peritoneally injected) thrice per day, over a course of 7 days consecutively, reportedly
induced: (1) massive destruction of cerebellar myelin (determined via immunohistochemical
staining); (2) axonal integrity losses; (3) inflammatory cell recruitment; and (4) mild reactive
astrogliosis (evidenced through glial fibrillary-acidic protein (GFAP) staining); as well as (5)
functional motor deficits including: hindlimb paralyses, tremors, and even body weight
reductions (Buch et al., 2005). Minor limitations of the study of Buch et al., may be that
researchers did not specifically quantify the approximate amount of oligodendrocyte ablation
with the MOG-iDTR line, and that they were unable to explicitly confirm that the apoptosis they
detected in cerebellar granular layers via fluorescein-12-terminal deoxynucleotidyl transferase
dUTP nick end labeling (TUNEL) staining was indeed from oligodendrocytes, and not granule
cells (Buch et al., 2005; Mathis et al., 2003).
Transgenic, cell-specific DTR expression has similarly shown promise as an effective ablation
model for other immune cells of mice, such as CD11c dendritic cells (DCs) and epidermal
Langerhans cells (LCs) (reviewed in Bennet and Clausen, 2007). More recently, the DTR
22
ablation system has been used to target Pet-1 expressing, 5-HT serotonergic neurons in a mouse
model to study the role of 5-HT neurons in thermoregulation, and particularly to compare
functional deficits of adult loss of 5HT-neurons vs those observed in 5-HT developmental
knockout mice (Lmx1b(f/f/p)) (Cerpa et al., 2014). The above study reported that a total dose of
as low as 1.5-2µg of DT, administered systemically (50 μg/kg DT once a week for 2–3 weeks)
was sufficient to selectively ablate approximately 80% of 5-HT neurons in the ventrolateral
medulla and, the raphe nuclei of the medulla oblongata (Cerpa et al., 2014). One limitation of
the work of Cerpa et al., could arguably be that they were apparently unable to determine a DT
dosage and schedule that could effectively ablate approximately 99-100% of 5-HT neurons
without incurring unwanted side effects (sickliness, weight loss, mortality etc.), even in control
wild-type (WT) animals (Cerpa et al., 2014).
Despite the potential as a cell-specific suicide gene tool displayed by the DTR ablation system in
the aforementioned studies, the system is not without severe drawbacks and limitations, foremost
of which is its inherent species limitation. Most human and other primate cells express HB-EGF,
the cell-surface receptor for DT endocytosis, and thus the DTR ablation system cannot be used to
selectively target any specific primate cell type (Fen et al., 1993; Vaughan et al., 1992).
Additionally, there exists the possibility for minor bystander effects to occur with the use of DTR
ablation, as there has been evidence presented that the HB-EGF receptor may not be vital for the
entry of DT into, but may instead only vastly accelerate the rate of DT uptake via endocytosis
(10-50% toxin uptake/minute with the receptor vs. 1-2% uptake/minute without the receptor), as
the toxin can apparently still enter cells through interaction with clathrin-coated pits which
enable it to be endocytosed during a bulk turnover of cell-surface proteins (Almond and Eidels,
1994). Although the slower, alternative entry of DT may not initially appear entirely relevant,
only a single DT molecule is necessary to induce cell death, and thus the possibility for a
bystander effect with DTR ablation is nevertheless is a serious concern (Yamaizumi et al., 1978).
Finally, and most significantly for any researcher considering the use of the DTR ablation system
for selective cell-killing, the obvious issue of toxicity to the user in the event of any emergency
(accidental self injection etc.) is the most vital concern- as 0.1µg/kg body mass is fatal for a
human user (Pappenheimer et al., 1982).
23
3.1.2 Herpes-Simplex Virus Thymidine Kinase and Ganciclovir
Gene-directed enzyme-prodrug therapies (GDEPTs) represent an umbrella category for a
variety of cell-ablation strategies (reviewed in Zhang et al., 2015). The combination of the
herpes-simplex virus thymidine kinase 1 (HSV-TK/HSV-TK 1), transgenically expressed in a
cell-type designated for ablation and one of its specific substrates- namely ganciclovir (GCV, or
9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl] guanine), a deoxyguanosine (purine
nucleoside) analog, has been demonstrated to be an effective method of targeted ablation, both in
anti-cancer and regeneration studies (reviewed in Zhang et al., 2015; Chou and Hong, 2014;
Ikeshima-Kataoka and Yuasa, 2008; Balzarini et al., 1993; Oliver et al., 1985; Nishiyama and
Rapp, 1979).
The mechanism of GCV-mediated cytotoxicity/ablation occurs in several steps: (1)
phosphorylation of GCV first to GCV-monophosphate by HSV-TK (or other viral thymidine
kinases, such as that of cytomegalovirus), and subsequently to GCV-triphosphate via
endogenous cellular kinases in two steps; (2) incorporation of GCV-triphosphate into cellular
DNA strands during replication, inducing chain termination- as GCV-triphosphate is a weak
substrate for elongation; and finally – (3) apoptosis, due to the aforementioned failure of DNA
replication (reviewed in Bagó et al., 2016; reviewed in Zhang et al., 2015; Balzarini et al., 1993;
Beltinger et al., 1999; Fischer et al., 2005). HSV-TK is understood to have approximately 1000-
fold greater affinity for the initial phosphorylation step than endogenous cellular thymidine
kinases, preventing general cytotoxicity in non-HSV-TK expressing cells at concentrations of
GCV lethal to cells expressing the viral thymidine kinase (reviewed in Bagó et al., 2016;
reviewed in Zhang et al., 2015; Balzarini et al., 1993).
Investigations into the precise apoptotic pathway involved in HSV-TK and ganciclovir mediated
ablation determined that the enzyme-prodrug combination first initiates expression of a cell death
receptor- “the first apoptosis signal receptor” (Fas/FasR; also referred to as cluster of
differentiation 95 (CD95)) and p53- which translocates CD95 to the cell outer-surface, where a
cell-death-inducing signalling complex (DISC), composed of Fas-associated death domain
protein (FADD) and caspase-8, is formed (Beltinger et al., 1999). The previous results showing
a p53 and CD95-dependent mechanism of apoptosis have been controversial, as further studies
demonstrated that in certain cell types, such as glioma cells, the HSV-TK and ganciclovir
24
apoptotic process occurs independently of p53 or death receptor expression, and instead involves
the down-regulation of expression of B-cell lymphoma 2 (Bcl-2) proteins, known apoptotic
regulators, involved as part of a series of separate mitochondrial pathways (Fischer et al., 2005).
Perhaps the most critical drawback of the HSV-TK and GCV cell ablation system is the presence
of a significant “bystander effect,” referring to the lack of complete selectivity of the GDEPT to
only target HSV-TK-expressing cells for apoptosis (reviewed in van Dillen et al., 2002; reviewed
in Mesnil et al., 2000). While the presence/expression of the viral thymidine kinase is required
for significant phosphorylation of GCV to GCV-monophosphate, and initiation of the latter two
cellular-kinase mediated phosphorylations to produce the active, DNA replication-chain
termination and apoptosis-inducing form of GCV-triphosphate, apoptosis can still occur in cells
neighbouring HSV-TK expressing cells, via the diffusion of GCV-monophosphate (as well as
GCV-di and triphosphate to a lesser degree) across cell membranes, a process mediated by gap
junction intercellular channels (GJICs), particularly connexin 43 (Cx43) (Asklund et al., 2003;
Burrows et al., 2002; Dilber et al., 1997).
Such a bystander effect mechanism has shown not to be universally homogenous among cell
types/cell lines, as some cells, such as those of the rat colon, DHD/K12/TRb cell line, have been
demonstrated to retain a bystander effect even when cultured in lower densities without physical
contact, or when treated with blockers of GJICs, such as 18 alpha-glycyrrhetinic acid or 1-
octanol; the retention of a bystander effect in DHD/K12 cells has been suggested to occur due to
the presence of a soluble factor encapsulating GCV-triphosphate, allowing its diffusion through
neighbouring cells, independent of the presence of Cx43 or other GJICs (Princen et al., 1999).
The soluble factor that may allow a gap-junction independent bystander effect to occur for
certain cell types has been suggested to be apoptotic vesicles, as evidenced by electron
microscopy studies (Freeman et al., 1993). In the study of gliomas, dexamethasone has been
indicated as an additional inhibitor of the HSV-TK/GCV ablation system’s bystander effect, via
the downregulation of Cx43 expression, resulting in: (1) reduced GJIC-mediated cell-cell
communication; in addition to (2) growth inhibition; (3) incorporation of thymidine; and (3)
apoptotic cascade modulation- a process accomplished through reducing CD95 expression, and
increasing expression of the two anti-apoptotic proteins, cellular inhibitor of apoptosis protein
2/baculoviral IAP repeat-containing protein3 (cIAP2/BIRC3) and B-cell lymphoma-extra large
25
(Bcl-xL), which interfere with caspases and prevent cytochrome c release respectively (Robe et
al., 2005).
Previous research has displayed ablation success with the HSV-TK and ganciclovir GDEPT for a
variety of cancer cell lines, including: murine lymphomas, murine mammary carcinomas, and
murine sarcomas (Moolten et al., 1990; Balzarini et al., 1993; Moolten, 1986). The range of
animal cancer models in which the HSV-TK and ganciclovir system has been employed further
range from leukemia, glioma, liver cancer, bladder cancer, oral cancer, and adenocarcinoma
(reviewed in Zhang et al., 2015; Bondanza et al., 2011; Ribot et al., 2011; Staquicini et al., 2011;
Kakinoki et al., 2010; Tang et al., 2009; Greish et al., 2010; Chen et al., 2010; Ambade et al.,
2010).
In regards to the ablation of neural cell types, the HSV-TK/GCV system has been utilized to
ablate rat glioma cells (C6 cell line), as well as murine cerebellar neural stems cells (C17.2 cell
line) (Li et al., 2005; Pu et al., 2011). For C17.2 cells, using the MTT colorimetric, cell-
metabolic activity and viability assay- wherein the activity of NAD(P)H-dependent cellular
oxidoreductase enzymes to reduce the tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT), to the insoluble, purple-coloured formazan is employed as
a metric of cell viability, a final concentration of 1µg/mL of GCV was determined to be
sufficient to yield approximately 99% ablation of HSV-TK expressing C17.2 cells after a time
period of 7 days (Pu et al., 2011). In both the studies of C6, and C17.2 cells, a strong bystander
effect was detected, whereby mixed HSV-TK-expressing to endogenous (non-HSV-TK-
expressing) cell ratios of 1:16 were sufficient to yield ablation of approximately 25% of all cells
(Li et al., 2005; Pu et al., 2011).
A second study that evaluated HSV-TK/GCV ablation as well as bystander effect with C17.2
cells showed that 25µM was sufficient to kill ~95% of HSV-TK-expressing cells after 48hrs of
treatment, and when grown in mixed populations with two non HSV-TK-expressing, glioma cell
lines (U87-MG and LN-18), the bystander effects were identified to be cell-type dependent, with
one apoptotic HSV-TK+ C17.2 cell accompanying ten U87-MG cells, or five LN-18 cells (Uhl et
al., 2005). A third study involving the use of the HSV-TK/GCV ablation system showed nearly
100% death of human-ESC-derived, Ki67+ NPCs with 40µM (~10µg/mL) GCV treatment after
200hrs, and virtually no toxicity on control NPCs in a separate population (Tieng et al., 2016).
26
The HSV-TK+GCV ablation system has additionally been previously utilized to selectively kill
murine astrocytes in the central, as well as peripheral and enteric nervous systems using a GFAP
promoter (Sofroniew et al., 1999; Bush et al., 1999; Bush et al., 1998). Through subcutaneous
osmotic minipump administration of 100mg/kg/day of GCV for a period of fourteen days, one
study demonstrated the selective ablation of GFAP-expressing astrocytes of the small intesntinal
jejunum and ileum and subsequent development of fulminant jejuno-ileitis (Bush et al., 1998). A
year later, Bush et al. similarly displayed the targeted cell-killing of reactive astrocytes in
forebrain-stab-injured mice, with a GCV dose of 100mg/kg/day administered for one week,
which similar to experiments attempting to prevent glial scar formation in SCI with STAT3
knockouts, led to increased immune cell (CD45+ leukocyte) infiltration, and blood-brain-barrier
repair failure, yet also increased neurite outgrowth (Bush et al., 1999; Herrmann et al., 2008).
In addition to astrocytes, definitive neural stem cells (dNSCs) have additionally been selectively
ablated with a GFAP promoter (Sachewsky et al., 2014; Imura et al., 2003; Morshead et al.,
2003). dNSCs, as separate from primitive, Oct-4-expressing, neural stem cells (pNSCs) were
identified partly through work that demonstrated that GCV treatment could selectively kill only
adult and postnatal, yet not early embryonic (pre-12.5 days) GFAP-expressing NSCs of the
murine CNS (Imura et al., 2003). Research from the same year as Imura et al. additionally used
GCV to ablate GFAP-expressing, dNSCs, and resultantly showed that while GFAP-expressing
dNSCs are present in the murine forebrain, they are not found in the retina (Morshead et al.,
2003).
A more recent study employing a similar, GFAP-promoter-driven HSV-TK transgenic mouse
model confirmed the presence of a rare population of adult, Oct-4-expressing (and GFAP-non-
expressing) pNSCs (AdpNSCs) in the mouse forebrain peri-ventricular region via the GCV-
mediated selective ablation of GFAP+ dNSCs (Sachewsky et al., 2014). Sachewsky et al.
additionally discovered that GCV can remain stable and still induce cell death of HSV-TK-
expressing, GFAP+, adult murine dNSCs as late as 10 days after an initial 16-hour treatment
(Sachewsky et al., 2014). In regards to SCI model usages, the HSV-TK+GCV ablation system
has been used in mice to successfully target both endogenous neuron-glial antigen 2+ (NG2+)
pericytes and OPCs, as well as endogenous GFAP+ reactive astrocytes (Hesp et al., 2018;
Faulkner et al., 2004).
27
As the HSV-TK/GCV ablation system’s mechanism of inducing apoptosis requires incorporation
of GCV-triphosphate into replicating DNA to prevent chain-elongation, it would be natural to
assume at first glance that an additional limitation of the system would be an inability to target
all non-dividing, post-mitotic or senescent cells; however, such a limitation has been
demonstrated to be false (Tieng et al., 2016; Laberge et al., 2013; Beltinger et al., 2000).
Cellular and molecular studies have determined that the principal mechanism of driving
apoptosis in the HSV-TK/GCV combination is in fact the disruption of mitochondrial DNA
synthesis, rather than genomic/nuclear DNA synthesis, and as many post-mitotic cells, such as
mature neurons, still undergo mitochondrial turnover, they still remain potential targets for HSV-
TK/GCV (Tieng et al., 2016; Laberge et al., 2013; Beltinger et al., 2000). After the GCV-
triphosphate-mediated interruption of mitochondrial DNA replication, it has been demonstrated
that affected cells (e.g., GCV-treated, HSV-TK-expressing, senescent human fibroblasts) suffer
mitochondrial membrane integrity losses, followed by cytochrome c release from the
mitochondria into the cytosol, and eventually caspase activation and nuclear fragmentation
(Laberge et al., 2013; Beltinger et al., 2000).
The HSV-TK/GCV GDEPT has certainly demonstrated promise as an effective and efficient
method of cell ablation in vitro and in vivo, as mentioned previously in numerous animal models
of cancer, and a few regeneration models (reviewed in Zhang et al., 2015; Ikeshima-Kataoka and
Yuasa, 2008). The bystander effect inherent in HSV-TK/GCV ablation, useful in cancer-based
utilizations for the ability to target non-transfected tumour cells, remains its principal detractor
for studies of regeneration, in particular for traumatic SCI models to selectively target neurons or
oligodendrocytes derived from transplanted NPCs (reviewed in Zhang et al., 2015; reviewed in
Ahuja et al., 2017b).
3.1.3 Herpes-Simplex Virus Thymidine Kinase and Brivudine
One option for mitigating the aforementioned bystander effect in HSV-TK/GCV ablation, is to
pair the viral enzyme with an alternative prodrug, particularly a pyrimidine, rather than purine
analog (reviewed in Dachs et al., 2009; reviewed in Degrève et al., 1999). Pyrimidine analogs,
such as (E)-5-(2-bromovinyl)-2'-deoxyuridine, also known as brivudine (BVDU), unlike purine
analogs (e.g., GCV), require the activity of HSV-TK or another viral thymidine kinase for not
28
only the first, but also the second phosphorylation step towards producing their active, cytotoxic
triphosphate form (reviewed in Dachs et al., 2009).
As Cx43 and other gap junctions are less permeable to diphosphate forms of pyrimidine (or
purine) analogs than to monophosphate forms, smaller quantities of the diphosphate form can
accumulate in neighbouring cells, resulting in a reduction in the number of neighbouring cells
that are able to accumulate the pyrimidine analog-triphosphate and undergo chain termination
during DNA replication and subsequently apoptose, than in respective neighbouring cells with
the use of a purine analog (reviewed in Dachs et al., 2009).
To compare the bystander effect of pyrimidine analogs, such as BVDU, with purine analogs,
such as GCV, one set of experiments using mixed populations of various ratios of HSV-TK+ and
HSV-TK- osteosarcoma cells found that: only by using a BVDU concentration of at least
250µM, one ablated HSV-TK+ cell was found to be capable of killing only one additional
neighbouring HSV-TK- cell; whereas even by using a GCV dose of at least 50µM, one ablated
HSV-TK+ cell was found to be capable of killing between seven to thirteen additional
neighbouring HSV-TK- cells (reviewed in Degrève et al., 1999). Such a reduced bystander
effect, although perhaps detrimental for some cancer-based uses, would appear on the surface to
make pyrimidine analog prodrugs, namely BVDU, sufficiently more useful for more selective
ablation approaches in SCI and other neuroregeneration studies.
The inhibitory effects of brivudine on the replication of human herpes simplex virus 1 (HSV-1)
have been understood since the 1980s, and were swiftly harnessed towards cell ablation methods
using transgenic/HSV-TK-expressing cells, primarily in cancer studies, such as those with
murine models of mammary carcinoma and, leukemia, in which brivudine was found to induce
significant apoptosis of HSV-TK-expressing cells at concentrations ranging from 5000-20,000
times below the equivalent requirements for wild type cytotoxicity (reviewed in De Clercq,
2005; reviewed in De Clercq, 2004; Balzarini et al., 1985a; Balzarini et al., 1985b; Balzarini et
al., 1985c; Balzarini et al., 1986; Balzarini et al., 1989; Balzarini et al., 1993).
Mechanistically, it has been elucidated that in contrast to purine analogs, such as GCV, the
primary target of BVDU in its cytotoxic effects on HSV-TK-expressing cells is thymidylate
synthase, rather than purely DNA incorporation during replication (Balzarini et al., 1987). One
ablation study involving murine mammary carcinoma cells transgenically expressing HSV-TK
29
along with rigorous exploration of intracellular biochemical pathways demonstrated thymidylate
synthase to be the principal BVDU target via showing that: a) cytotoxic effects could be more
easily reversed via the addition of deoxythymidine (dThd) than 2(1)-deoxyuridine (dUrd); b)
BVDU had a greater effect on preventing dUrd incorporation than dThd incorporation into
replicating cellular DNA; and, c) BVDU exhibited a strong inhibitory effect on the pathway
involved in incorporating 2'-deoxycytidine (dCyd) into cellular DNA thymidylate (Balzarini et
al., 1987). In regards to apoptosis, it has been determined that BVDU initiates the process via
activation of the c-Jun/activator protein-1 and Fas ligand/caspase-8 pathway, as opposed to the
Fas-ligand-independent pathway initiated by GCV (reviewed in De Clercq, 2005; Tomicic et al.,
2003). Although not explicitly determined, it is hypothesized that BVDU, like GCV, also
activates the mitochondrial damage pathway involving a caspase-mediated cleavage of Bcl-2
(reviewed in De Clercq, 2005; Tomicic et al., 2003).
The optimal concentration of BVDU necessary to induce effective total cell ablation (arbitrarily
defined hereafter as ~90% in under 5 days) is not perfectly understood but is known to be cell
type dependent (Balzarini et al., 1985 (3)). For murine mammary carcinoma (FM3A) HSV-TK+
cells, the 50% inhibitory dose/concentration (of viral plaque formation- ID50/IC50 for tumor cells
inhibition) BVDU dose has been shown to be 0.5ng/mL, versus 11µg/mL for normal (non-HSV-
TK expressing) FM3A cells (Balzarini et al., 1985 (3)).
In contrast, the equivalent BVDU ID50 was found to be: (1) between 0.01-0.02µM (~0.00333-
~0.00666 µg/mL) for primary rabbit testes (PRT) cells; (2) between 0.02-0.06 µM (~0.00666-
~0.020 µg/mL) for MDA-MB-435 human breast carcinoma cells; (3) between 0.045-0.105 µM
(0.015-0.035 µg/mL) for rat gliosarcoma (9L) cells; and (4) as low as 0.01 µg/mL for baby
hamster kidney 21 (BHK-21) cells (as evaluated in a plaque reduction test of plated monolayers)
(Reefschläger et al., 1982; Grignet-Debrus et al., 2000; Field et al., 1984). For osteosarcoma
cells, previous work determined the IC50 for BVDU to be 0.035µM (0.012µg/mL) for HSV-TK-
expressing cells vs. ~900µM (~300µg/mL) for HSV-TK negative/normal tumor cells (reviewed
in Degrève et al., 1999).
BVDU uses in relation to neural cells appear to be rarer, however most principally it has shown
success in treating herpetic encephalitis, wherein infected neurons contain viral (as opposed to
transgenic, cellular-driven) HSV-TK expression (reviewed in Rosato and Leib, 2015; Wigdahl et
30
al., 1984; Wigdahl et al., 1983). In one of the aforementioned studies of BVDU’s effects on
treating encephalitis, a dosage of 10µg/mL was shown to produce near 100% elimination of HSV
plaques after 3 days in human fetal DRG neurons (Wigdahl et al., 1984).
Similar to GCV, BVDU has most certainly shown potential as an efficient prodrug for inducing
cell ablation in combination with transgenic HSV-TK expression. Although it has often been
overshadowed by GCV in cancer research, due to its reduced bystander effect limiting its ability
to target neighbouring, non-HSV-TK-expressing tumor cells, this very same flaw could
theoretically represent a significant strength in regards to its use in studies of neuroregeneration,
such as targeting transplanted NPC-derived cells in traumatic SCI models, due to its enhanced
selectivity over GCV.
3.1.4 E. coli Nitroreductase (NfSA/NfsB) and CB1954
The combination of the E. coli oxygen-insensitive NAD(P)H, flavin mononucleotide (FMN)-
containing nitroreductase enzyme (either NfSA- 240 amino acids (a.a.), or NfsB- 217a.a.;
hereafter referred to as “NTR”) with the nitroaromatic alkylating agent prodrug, CB1954 [5-
(aziridin-1-yl)-2,4-dinitrobenzamide], is a GDEPT that has demonstrated considerable promise
in both cancer and regeneration research for over two decades (reviewed in Zhang et al., 2015;
reviewed in Searle et al., 2004; reviewed in Denny 2002; reviewed in Williams et al., 2015; Vass
et al., 2009; Drabek et al., 1997; Clark et al., 1997). CB1954 was first identified for the purpose
of tumor cell ablation in the late 1960s due to its cytotoxic potency against the Chester Beatty
Walker rat carcinoma cell line, WS, during screens of various nitrophenylaziridine compounds
(reviewed in Knox et al., 2003; reviewed in Knox et al., 1993; reviewed in Workman et al.,
1986; Connors and Melzack, 1971; Cobb et al., 1969; Khan and Ross 1969). The sensitivity of
Walker rat carcinoma cells to CB1954 was later identified to be due to the expression of NQO1
(DT-diaphorase), a similar type-I (oxygen-independent) nitroreductase enzyme to NfsA/NfsB
(reviewed in Knox et al., 2003). In contrast to rat tumors, human tumors were indentified to be
largely insensitive to CB1954 due to the severely reduced CB1954-catalyzing efficiency of the
human form of NQO1 (reviewed in Knox et al., 2003).
Mechanistically, type-I nitroreductases, such as NTR and NQO1, catalyze a two-electron transfer
to CB1954, using NADH or nicotinamide adenine dinucleotide phosphate (NADPH) as an
intermediate substrate, and reduce the 4-nitro group of CB1954 to a 4-hydroxylamine, which
31
subsequently undergoes a non-enzymatic reaction with a thioester (e.g., acetyl coenzyme A)
producing a difunctional, DNA-interstrand crosslinking agent (reviewed in Williams et al., 2015;
reviewed in Knox et al., 2003; reviewed in Knox et al., 1993; Knox et al., 1988; Roberts et al.,
1986; Connors and Melzack, 1971). After DNA interstrand crosslinking from the reduced form
of CB1954, affected (NTR-expressing) cells are understood to undergo apoptosis in a p53-
independent manner (Cui et al., 1999).
In regards to applications (both in vitro and in vivo), the NTR/CB1954 ablation system has been
used to target and successfully kill mouse fibroblasts in vitro using a concentration of 20µM with
no toxicity observed in control mouse fibroblasts below 500 µM, as well as CD2-promoter-
driven-NTR-expressing mouse T cells in an in vivo setting, with no significant systemic toxicity
(Drabek et al., 1997). Similarly, NTR/CB1954 has demonstrated similar selectivity and
ablational efficiency in targeting mouse mammary gland luminary cells using ovine beta-
lactoglobulin promoter-driven NTR expression, as well as with mouse pre-adipocytes (Clark et
al., 1997; Felmer and Clark, 2004). With NTR-expressing mouse fibroblast NIH3T3 cells, one
study reported approximately 99% ablation using a treatment of 0.1mM CB1954 for an exposure
period of only 24 hours (Bridgewater et al., 1995).
Further evidence has demonstrated CB1954’s ablational efficiency against human cell types
genetically engineered to express NTR, such as the LS174T colorectal cancer cell line, or the
SUIT2, BxPC3, and AsPC1 pancreatic cancer cell lines, which have been shown to be as much
as 500 times more sensitive (in terms of apoptosis and cell killing) to CB1954 treatment than
their respective control parent lines (Green et al., 1997). Additionally, in human ovarian
carcinoma cells of the cell lines SK-OV-3 or IGROV-1, transgenic NTR expression has been
shown to increase CB1954 sensitivity by up to 2,000-fold over control parent line cells (Weedon
et al., 2000).
Attempts have been made to further improve the sensitivity of NTR-expressing cells to CB1954
treatment, such as one using six mutant forms of NTR each with amino acid substitutions, which
identified one specific mutant NTR variant with an amino acid substitution at residue F124 that
conferred a 5-fold increase in CB1954 sensitivity in human SK-OV-3 ovarian carcinoma cells
(Grove et al., 2003). A follow up study examining double mutants of NTR identified an even
more CB1954-sensitive variant than the F124-NTR mutant, which rendered SK-OV3 cells up to
32
14-17 times more sensitive to CB1954 ablation treatment than their regular (NfsB) NTR-
expressing counterparts (Jaberipour et al., 2010). Additional optimizations of NTR/CB1954
ablation have reported improvements in ablational efficiency/prodrug sensitivity by: (1) using
alternative forms of NTR, such as using the other oxygen-insensitive E. coli nitroreductase,
NfsA, which was demonstrated to be 10 times more efficient in the catalytic efficiency of
CB1954 reduction; or (2) by developing a human-codon preference-adapted version of the NfsB
gene using 144 silent base changes to enhance NTR transcription and subsequently translation in
human cell types, which reportedly rendered several human cell lines up to 10 times more
sensitive to CB1954 ablation than their regular NTR/NfsB counterparts (Prosser et al., 2010;
Grohmann et al., 2009).
Relatively few neural uses of the NTR/CB1954 have been applied, however one study using the
nestin promoter to express NTR in neonatal rat cerebellar NPCs in an in vivo setting reported
significant cerebellar degeneration and volume reduction, in addition to ataxia as well as
sensorimotor functional losses after three days of daily intracerebellar treatment with 0.5mg/kg
CB1954, starting at the five-day age point (Kwak et al., 2007). Using 20µL
intracerebroventricular injections of 2mg/mL of CB1954 once daily for a period of 2 days, the
same study additionally reported significant degeneration in the hilus and granule cell layer of
the hippocampal dentate gyrus of similar nestin promoter-driven, NTR-expressing transgenic
neonatal rats (Kwak et al., 2007). CB1954 reportedly showed no relevant toxicity for control,
non-NTR-expressing animals in either neonatal rat NPC ablation experiment (Kwak et al., 2007).
The NTR/CB1954 enzyme-prodrug combination is not without its drawbacks, as similar to the
HSV-TK/GCV cell ablation system, it has been shown to possess a moderate, yet significant
bystander effect (Felmer and Clark 2004; Green et al., 1997; Bridgewater et al., 1997). The
bystander effect for NTR/CB1954 is understood to occur due to the presence of at least two cell
permeable metabolites, specifically the reduced forms of CB1954 possessing the 4-
hydroxylamine group, both before and after thioester activation (Bridgewater et al., 1997). In
practical application, the bystander effect of NTR/CB1954 has been demonstrated to be cell
density and type dependent, and for the specific instances of human pancreatic (SUIT2, BxPC3,
and AsPC1) as well as human colorectal (LS174T) cancer cell lines, a bystander effect was
detected by only 10% of NTR-expressing cells in one study (Green et al., 1997; Grove et al.,
1999).
33
Overall, the NTR/CB1954 enzyme-prodrug ablation system has been demonstrated to be a
promising cellular/molecular tool for selective cell ablation (reviewed in Zhang et al., 2015;
reviewed in Williams et al., 2015; reviewed in Denny, 2002). The primary hurdle with regards
to its use in neuroregeneration studies, such as those to examine the restorative functions of
transplanted NPCs (and derived cells, such as oligodendrocytes and various types of neurons) at
different timepoints after spinal cord injury, is of course the system’s moderate, yet significant
bystander effect, which could inevitably result in unintended abnormalities and inabilities to
distinguish the specific repair brought on by the transplanted NPCs (Bridgewater et al., 1997).
Nevertheless, the NTR/CB1954 enzyme-prodrug combination for cell ablation still represents a
more promising alternative than species-restricted options such as transgenic-diptheria toxin
receptor expression, previously stated to be unusable for the selective ablation of human cell
types (reviewed in Zhang et al., 2015; Bennett and Clausen, 2007).
3.1.5 E. coli Nitroreductase (NfsB/NTR) & Metronidazole (Mtz)
Building on the success of the NTR/CB1954 enzyme prodrug cell ablation system, other
prodrugs have been evaluated in combination with NTR for a variety of purposes, including
finding a prodrug with a reduced bystander effect to improve the specificity of NTR cell ablation
in the areas of regeneration and development studies (reviewed in Zhang et al., 2015; reviewed
in Williams et al., 2015; reviewed in Dachs et al., 2009; reviewed in Denny 2002). One specific
prodrug identified to possess a reduced bystander effect in combination with NTR/NfsB is the
nitroimidazole anti-protozoan compound, metronidazole (Mtz) (reviewed in Zhang et al., 2015;
reviewed in Williams et al., 2015; reviewed in White and Mumm, 2013; reviewed in Curado et
al., 2008; Bailey et al., 1996).
The NTR/Mtz combination reportedly offers a plethora of benefits over other cell ablation
systems/enzyme-prodrug combinations, including: (1) increased efficacy; (2) greater specificity;
(3) resistance to promoter leakiness (e.g., off-target expression of the enzyme in non-target cell
types); and (4) quicker inducibility; in addition to (5) the ability to efficiently target non-
proliferating cells (reviewed in Curado et al., 2008; Curado et al., 2007; Pisharath et al., 2007).
The use of Mtz with NTR has specifically been described as “cell-type specific, inducible,
reversible, rapid and scaleable” (reviewed in Curado et al., 2008). The mechanism of ablation
induced in NTR-expressing cells by Mtz is understood to be largely similar, if not nearly
34
identical to that of the NTR/CB1954 combination, whereby NADH/NADPH first reduces NTR
itself, then subsequently NTR catalyzes the reduction of the non-cyclic nitrogen of Mtz,
producing a cytotoxic radical-containing product that acts as a DNA-interstrand crosslinking
agent (reviewed in Curado et al., 2008; Edwards, 1993; Lindmark and Müller, 1976). The DNA-
crosslinking action of the activated/reduced form of Mtz then induces apoptosis in a p53-
independent mechanism (believed to be along the intrinsic/mitochondrial pathway), involving
caspase releases, plasma-membrane integrity losses, and nuclear fragmentation (reviewed in
Curado et al., 2008; Cui et al., 1999; Edwards, 1993; Lindmark and Müller, 1976).
One of the first cell ablation usages of the NTR/Mtz ablation was to target human cytoxic T
lymphocytes (hCTLs) in an in vitro setting (Verdijk et al., 2004). At a concentration of 1mM,
Mtz was found to produce a 79% proliferation inhibition in hCTLs, and additionally was
reportedly toxic for both NTR-expressing and control hCTLs at higher concentrations (Verdijk et
al., 2004). In its first reported in vivo utilization, in the Danio rerio zebrafish model, NTR-
ubiquitously-expressing (under the EF1α promoter) embryos were treated with 7.5-10mM of
Mtz, inducing muscular necrosis, cardiac damage, and necrosis over a 24-hour timeframe (56
hours post-fertilization (hpf)-80hpf) (reviewed in Pisharath and Parsons, 2009; Pisharath et al.,
2007; Pisharath 2007).
In the same work of Pisharath et al. in 2007, a treatment of 10mM of Mtz for a period of 24
hours was found to completely ablate embryonic, NTR-expressing, zebrafish pancreatic beta
cells that had previously been developed to use the preproinsulin gene promoter (ins) to drive
expression of an NTR-mCherry (a red fluorescent marker similar to red-fluorescent protein
(RFP)) fusion protein; the ablation of pancreatic beta cells was confirmed both by examining
fluorescence and insulin transcript levels (reviewed in Pisharath and Parsons, 2009; Pisharath et
al., 2007; Pisharath 2007). The very same Mtz treatment (10mM for 24 hours) was found to
have no off-target effects or toxicity in control zebrafish embryos, and additionally show no
apparent bystander effects on neighbouring cells of the developing exocrine pancreas, or in any
other endocrine pancreatic cells (e.g., alpha cells or delta cells, etc.) (reviewed in Pisharath and
Parsons, 2009; Pisharath et al., 2007; Pisharath 2007).
Subsequent in vivo examination of the NTR/Mtz system has included the successful complete
ablation: (1) of embryonic zebrafish cardiomyocytes (10mM for 24 hours); (2) embryonic
35
zebrafish hepatocytes (10mM for 72 hours); (3) zebrafish primordial germ cells (10mM for 48
hours); (4) zebrafish hoxa13a/hoxd13a-expressing fin-fold mesenchyme (20mM for 3 day); (5)
zebrafish podocytes (10mM for 12 hours); (6) zebrafish xanthophores (10mM for 24 hours); as
well as (7) a reproduction of the targeting of zebrafish pancreatic beta cells (5mM for 12 hours)
(reviewed in Curado et al., 2008; Curado et al., 2007; Zhou et al. 2018; Lalonde and Akimenko,
2018; Huang et al., 2013; Zhou and Hildebrandt, 2012; Walderich et al., 2016). Upon removal
of Mtz, several embryonic zebrafish cell types have been shown to regenerate, such as zebrafish
pancreatic beta cells after 36 hours, and fin-fold mesenchyme after only 48 hours (Pisharath et
al., 2007; Lalonde and Akimenko, 2018). The NTR/Mtz system has additionally been used for
the purpose of creating inducible infertility models of both male and female zebrafish, by
targeting NTR-expressing spermatagonia and oocytes respectively (Hsu et al., 2010a, Hsu et al.,
2010b).
In order to improve the ablational efficiency of the NTR/Mtz system, attempts have been made
to develop a form of the NTR (NfsB) enzyme with a greater catalytic efficiency for reducing Mtz
(or other nitrimidazole/dinitrobenzamide substrates) (Mathias et al., 2014). The most successful
reported attempt at developing an improved version of NTR involved the discovery of a triple-
mutant (T41Q/N71S/F124T) form of the enzyme that has been demonstrated to induce
significant (compared to regular WT-NfsB/NTR controls) Mtz-induced ablation in vivo in both:
a) zebrafish spinal motor neurons after only 4 hours of 10mM Mtz treatment (only slightly less
than 24 hours of 10mM Mtz treatment on WT-NTR+ cells); and b) zebrafish spinal interneurons
after only 4 hours of as low as 4mM Mtz treatment (comparable to 24 hours of 10mM Mtz
treatment on WT-NTR+ cells) (Mathias et al., 2014).
The very same triple mutant form of NTR even permitted efficient in vivo ablation of zebrafish
cranial motor neurons, a more internal/deeper cell type than spinal inter- or motor neurons, with
only 4-hour treatments with 10mM Mtz (comparable to 24 hours of 10mM Mtz treatment on
WT-NTR+ cells) (Mathias et al., 2014). Taken together, the triple mutant form of NTR has
shown to be an enticing option for improving the ablation efficiency of the NTR/Mtz system
(Mathias et al., 2014).
In addition to zebrafish spinal motor and interneurons as well as cranial motor neurons, a
plethora of other neural cell types have been assessed in regards to NTR/Mtz ablation.
36
Additional neural cell types successfully ablated using the NTR/Mtz system in a zebrafish in vivo
setting include: (a) brachiomotor neurons (significantly reducing food intake); (b) dopaminergic
neurons (wherein 24h of 5mM Mtz followed by 48h of 7.5mM Mtz induced caspase 3 release,
ablation, and sensorimotor deficits); (c) radial glial cells (wherein 1h of 5mM Mtz treatment
applied daily for 3 days induced significant apoptosis in GFAP-positive telencephalic cells); and
(d) rod photoreceptors (complete ablation after 24h of 10mM Mtz treatment with no damage to
neighbouring cones) (Allen et al., 2017; Mathias et al., 2014; Godoy et al., 2015; Shimizu et al.,
2015; Montgomery et al., 2010). Remyelination mechanisms have similarly been studied in a
zebrafish model, as evidenced by a study that used NTR/Mtz to induce demyelination, via total
ablation of CNS oligodendrocytes after 48 hours of 10mM Mtz treatment, but yet observed
complete remyelination after 7 days of prodrug removal (Chung et al., 2013).
In addition to zebrafish, other model organisms that have been used with the NTR/Mtz system
have included: (1) Oryzias latipes, more commonly known as Medaka or the Japanese rice fish,
with which osteoblasts and pancreatic beta cells have been among target cell types successfully
ablated; and (2) Xenopus Laevis, more commonly known as the African clawed frog, with which
rods and oligodendrocytes have been selectively ablated (Willems et al., 2012; Otsuka and
Takeda, 2017; Choi et al., 2011; Kaya et al., 2012). As in zebrafish, retinal degeneration has
been studied in the frog model using the NTR/Mtz system to target rod photoreceptors (Choi et
al., 2011). Although in the work of Coi et al., cone degeneration occurred subsequently after rod
ablation, it is not understood to have occurred through any bystander effect of NTR/Mtz, but
rather through as a separate result of the process of retinal degeneration, as NTR expression was
controlled by the rhodopsin promoter, not found in cones (Choi et al., 2011).
Similar to the 2013 work of Chung et al., remyelination has been investigated in frogs, using
MBP-promoter-driven expression of NTR in mature oligodendrocytes followed by Mtz treatment
at the tadpole stage (Sekizar et al., 2015; Kaya et al. 2012). Sekizar et al. monitored cell division
and regeneration after removal of Mtz and suggested based on their findings that optic nerve-
residing, Sox10+ OPCs may be the source of the newly formed mature oligodendrocytes that
provided remyelination, and additionally that little replication of OPCs is necessary for
remyelination in Xenopus (Sekizar et al., 2015). Sekizar et al.’s findings indicate that one of the
critical differences in the capability of remyelination in amphibians versus mammals may be the
37
presence or absence respectively of sufficient quantities of endogenous OPCs in the optic nerve
(Sekizar et al., 2015).
Taken as a whole, the NTR/Mtz system has proven itself countless times as a simple and
efficient enzyme-prodrug combination for cell ablation (reviewed in Williams et al., 2015).
Despite the success of the NTR/Mtz, its usages have rarely extended beyond the fish and
amphibian model organisms, save for the 2004 work of Verdijk et al. using hCTLs in an in vitro
setting (reviewed in Williams et al., 2015; Verdijk et al., 2004). One of the only other known
mammalian usages of the NTR/Mtz consisted of ablating human lymphoma NB4 cells both in
vitro and in vivo transplanted in mice to generate tumors (McCormack et al., 2013). No
mammalian neural usages (in vitro or in vivo) of the NTR/Mtz system are known to have yet
occurred, however there is no obvious indication from the cellular and molecular aspects of the
system that such a feat should not be possible, nor that the system should intrinsically be limited
to neural cells of fish or amphibians (reviewed in Williams et al., 2015). As CB1954 has been
previously used to ablate NTR-expressing neural progenitors in rodents, it would seem feasible
that Mtz-mediated ablation should similarly be achievable in NPCs or NPC-derived cells (Kwak
et al., 2007).
Additionally, although the NTR/Mtz system has been demonstrated to lack any considerable
bystander effect, it still possesses drawbacks such as: (a) the availability of a suitable promoter
for expression in target cell types (a drawback seen in any gene-driven ablation system); (b) the
resistance of specific tissue/cell types to ablation (depending on the in vivo accessibility of the
cell type to the prodrug, regeneration capacity of the cell type, or ability of the cell type to
metabolize Mtz- as seen in hepatocytes); and (c) the requirement of longer treatments for certain
cell types depending on regenerative capacity among other factors (reviewed in Williams et al.,
2015; reviewed in Curado et al., 2008). The NTR/Mtz ablation system would appear to be an
ideal candidate choice for pursuing neuroregeneration loss/gain of function studies in rodent
models of SCI, most of all due to its lack of an observable bystander effect, however as
previously stated, the fact that no studies have yet pursued mammalian neural in vivo usages of
the system should nevertheless remain as a caution and caveat moving forward (reviewed in
Williams et al., 2015).
38
Cell Ablation
System
Advantages Disadvantages
Diptheria Toxin Receptor
(DTR)
-Minimal bystander effects
(though potential for one to exist)
-Unusable with human cells
(non-selective),
-general toxicity,
-extremely dangerous to user
HSV-TK+ Ganciclovir
(GCV)
-widely used/highly understood,
-high substrate affinity (GCV for
HSV-TK)
-no species restrictions
-Considerable bystander effects
-Potential difficulties ablating
non-proliferative/post-mitotic
cells
HSV-TK+Brivudine
(BVDU)
-Reduced bystander effects,
-reduced toxicity (over GCV),
-low carcinogenicity
-Minorly reduced BVDU
substrate affinity for HSV-TK
(vs. GCV)
-BVDU ablation is less widely
used/understood (vs. GCV)
-Potential difficulties ablating
non-proliferative/post-mitotic
cells
NTR+CB1954 -no species restrictions
-not reliant on cell division
-considerable bystander effects
NTR+Metronidazole
(Mtz)
-no species restrictions
-not reliant on cell division
-no known bystander effects
-highly proliferative cell types
may be slightly resistant
-potential difficulties with low
Mtz solubility
Table 2: An Overview of Cell Ablation Systems. A wide variety of gene-directed enzyme-
prodrug therapies (GDEPT) exist for achieving the goal of selective cell ablation, ranging from
transgenic expression of the diptheria toxin receptor (+administration of the toxin itself) for
use with rodent cells, to the herpes simplex virus thymidine kinase (HSV-TK) +GCV/BVDU
system, and the E. coli NfsB nitroreductase (NTR)+CB1954/metronidazole (Mtz) systems.
39
3.2 Gene Transfer Vectors
The first step towards creating lines of selectively ablatable NPCs for SCI neuroregeneration
studies is to successfully transfer the gene encoding an enzyme intended for use with an
apoptosis-inducing prodrug into the NPC genome (reviewed in Kantor et al., 2014; reviewed in
Manfredsson, 2014). In order to accomplish the task of NPC genomic transformation, a suitable
gene transfer vector must be selected. Two such vectors include: (1) lentiviruses; and (2)
transposons (reviewed in Kantor et al., 2014; reviewed in Manfredsson, 2014; reviewed in Joshi
et al., 2017).
3.2.1 Lentiviral Vectors
Lentiviruses (“slow-viruses” from the latin “lente”), which are composed of a dual copy,
positive sense single-stranded RNA genome covered by both a protein capsid and a lipid
envelope, are representatives of a genus of the retroviridae family (Baltimore Classification
Class VI) (reviewed in Sharon and Kamen, 2018). Genomic integration, a task accomplished by
the viral enzymes – reverse transcriptase (used for creating a DNA copy of the RNA viral
genome) and integrase (used for integrating the reverse transcribed viral DNA into the host cell
genome), has allowed lentiviruses to be developed as gene transfer vectors of transgenes
(reviewed in Sharon and Kamen, 2018).
While lentiviruses as gene transfer vectors offer such benefits as: (a) the ability to transfect cells
regardless of cell-cycle status (including transfecting post-mitotic cells); (b) the maintenance of
transgene expression in daughter cells (a feature not possessed by adenovirus/adeno-associated
viruses, as they are incapable of genomic integration of transgenes); (c) low immunogenicity;
and, (d) high transformation efficiency, they are not without considerable drawbacks (reviewed
in Sharon and Kamen, 2018; reviewed in Segura et al., 2013; reviewed in Durand and Cimarelli,
2011). Lentiviral vectors are unfortunately capable of inducing tumorigenicity due to proto-
oncogene activation, general insertional mutagenesis of target cells, and perhaps most critically
possess the risk, however minute, of a significant safety hazard (reviewed in Sharon and Kamen,
2018; reviewed in Durand and Cimarelli, 2011).
40
Most lentiviruses are derived from the exceptionally dangerous and incurable human
immunodeficiency virus 1 (HIV-1), although they have undergone considerable modifications,
deletions of protein coding genes, and mutations over three “generations” to minimize the risk
considerably (reviewed in Sharon and Kamen, 2018; reviewed in Stellberger et al., 2017). The
only remnants of HIV-1 in lentiviral vectors are the protein binding regions (involved in reverse
transcription and packaging), packaging signal (ψ), and long terminal repeat (LTR) (reviewed in
Sharon and Kamen, 2018). First generation lentiviruses, which were divided into a three plasmid
system consisting of: (1) a transfer plasmid; (2) a packaging plasmid; and, (3) an envelope
plasmid, were shown to possess both a large cloning capacity (~8.5 kilo-base pairs (kb)), and a
high transfection efficiency, but yet also too high a risk of recombination to produce pathogenic
replication competent lentiviruses (RCL) (reviewed in Sharon and Kamen, 2018; reviewed in
Schlimgen et al., 2016; reviewed in Cornetta et al., 2011).
In the next generation, the unneeded virulence factor genes virus protein U (vpu), viral
infectivity (vif), negative factor (nef), and virus protein R (vpr) were removed, in order to
attenuate any replication-competent virus produced, however rare (reviewed in Sharon and
Kamen, 2018). Finally in the third generation of lentiviral vectors, an additional plasmid was
added to the system, as the packaging plasmid was divided into two- one of which contained the
gene rev (encoding the Rev response element (RRE) mRNA transcript binding protein to allow
nuclear export), and the other of which contained a combination of the gene, group antigens
(gag), encoding a structural protein component of the virion’s capsid, nucleocapsid, and matrix)
and pol (encoding the reverse transcriptase and integrase) (reviewed in Sharon and Kamen, 2018;
Farley et al., 2015).
Additionally, in third generation lentiviruses the gene trans-activator (tat), encoding a
transcription-activating, 5’ LTR binding protein, was rendered unnecessary and removed, as the
5’ LTR-situated tat promoter was replaced with a CMV promoter (reviewed in Sharon and
Kamen, 2018). Self-inactivating (SIN) lentiviral vectors were additionally developed, in which
modifications were made to remove the promoter/enhancer regions of the viral 3’ LTR,
rendering the viral vector unable to perform complete RNA transcription (reviewed in Sharon
and Kamen, 2018; reviewed in Stellberger et al., 2017; reviewed in Segura et al., 2013). The
modifications employed to generate third generation lentiviruses enabled the risk of
recombination to be almost entirely eliminated (reviewed in Sharon and Kamen, 2018).
41
Lentiviral production generally consists of transiently transfecting human embryonic kidney
293T (HEK-293T) cells (or a similarly adherent cell type) through calcium phosphate
precipitation and harvesting the subsequently produced virus via collecting culture media
(reviewed in Sharon and Kamen, 2018). The harvested lentiviral culture media is then processed
through ultra-high-speed centrifugation (often above 90,000 g of relative centrifugal force
(RCF)), and virus pellets are resuspended at low volumes (several microlitres, depending on the
desired titer, starting culture media volume, time of transient transfection, density of adherent
cells used, etc.) and frozen at -80 degrees Celsius for long-term storage (reviewed in Sharon and
Kamen, 2018; Jiang et al., 2015). Although the titers of virus that can be produced through the
aforementioned method can be very efficient (up to 5x107 in unprocessed lentiviral media), the
serious drawbacks hindering the use of lentivirus are the length of time and labour involved in
production, high learning curve of production techniques with the possibility of failing to
generate sufficient titer (e.g., via accidentally suctioning off the viral pellet), and as mentioned
previously, the notorious, if actually minute, safety risks (if using first and second generation
lentiviral vectors specifically) (reviewed in Sharon and Kamen, 2018).
Overall, lentiviral vectors have demonstrated considerable success as gene transfer vehicles
(reviewed in Sharon and Kamen, 2018; reviewed in Stellberger et al., 2017). Although they
possess the beneficial properties of the ability to integrate within host cell genomes, the ability to
target both dividing and non-dividing cells, the ability to be passed on to daughter cells, and
strong transfection efficiency, lentiviral vectors are nevertheless hampered by the issues of
safety, as well as complexity and lengthiness in regards to production (reviewed in Sharon and
Kamen, 2018; reviewed in Stellberger et al., 2017).
3.2.2 Transposon Vectors
Non-viral vectors, such as transposons, are a generally more recent advancement in gene
transfer technology and offer a number of benefits over lentiviral vectors (reviewed in Vargas et
al., 2016; reviewed in Di Matteo et al., 2012). Some of these aforementioned benefits include:
(1) even lower immunogenicity, as the viral entry protein frequently used by lentiviral vectors,
vesicular stomatitis Indiana virus G-protein (VSV-G) can still incur an immune response from
some target cell types; (2) reduced development costs; (3) shorter production times, due to the
lack of additional required procedures such as transfecting adherent cells and spending additional
42
days collecting the produced virus particles, testing for RCL, and titrating the produced virus;
and perhaps most importantly, (4) increased safety, as transposon vector production does not
contain the rare, yet dangerous risk of recombination to form RCL that is inherent to lentiviral
production (reviewed in Vargas et al., 2016). Cellular uptake difficulty is one minor
disadvantage of transposon gene vectors, as unlike viral vectors, they cannot independently enter
target cells, and thus require an additional assistance method, such as electroporation (reviewed
in Vargas et al., 2016). The efficiency of cellular uptake and resultant transduction of transposon
vectors is known to be influenced by: (1) plasmid size; (2) cell type; and (3) transfection method
(reviewed in Vargas et al., 2016).
Transposons are simple mobile genetic elements, present in plants, animals, fungi and even some
bacteria, that either undergo: (a) a “copy and paste” mechanism, as in the case of Class I
retrotransposons (e.g., LTR-containing retrotransposons, long-interspersed nuclear elements
(LNES), and short-interspersed nuclear elements (SINES), etc.) wherein the transposable
element (TE)’s mRNA transcript is reverse transcribed back to DNA, then integrates elsewhere
in the host genome; or (b) a “cut and paste” mechanism, as in the case of Class II DNA
transposons, which utilize the enzyme, transposase to mediate their excision from a host genome,
prior to reinsertion at a new site (reviewed in Vargas et al., 2016; reviewed in Skipper et al.,
2013; reviewed in Di Matteo et al., 2012). Class II DNA transposons consist of the transposase
gene flanked by inverted terminal repeats (ITRs) that act as recognition sites for the transposase
enzyme (reviewed in Vargas et al., 2016; reviewed in Skipper et al., 2013).
As far as gene transfer vectors, Class II, “cut and paste” DNA transposons are the exclusive
choice used, in order to avoid excessive additional random insertions of a target gene (as usually
only one insertion is required in most procedures), yet some additional ones (depending on the
transposon vector used) are almost always unavoidable to some degree (reviewed in Vargas et
al., 2016; reviewed in Skipper et al., 2013; reviewed in Ivics et al., 2009). When used for gene
transfer, transposons are often, but not always, split into a two-plasmid system, with the first
plasmid containing the delivery gene of interest flanked by ITR recognition sites, and the second
plasmid containing the appropriate transposase gene (reviewed in Vargas et al., 2018). The two-
plasmid system reduces the chance of integration of the transposase gene itself, usually limiting
the number of integrations below an excessive/unwated amount (reviewed in Vargas et al.,
2018). Transposons and transposase enzymes are known to be self-regulatory, based on their
43
protein expression levels and transposon copy numbers (numbers of transposon genes present),
reducing activity at too high or low expression levels, thus unwanted excisions and integrations
are generally avoided with regards to gene transfer applications (reviewed in Vargas et al.,
2018). Two commonly used transposon vector systems are: (I) the piggyBac transposon; and
(II) the Tol2 transposon (reviewed in Vargas et al., 2016; reviewed in Ivics and Izsvák, 2010;
reviewed in Ivics et al., 2009; reviewed in Zhao et al., 2016).
The piggyBac transposon, previously known as IFP2, was originally discovered during
experiments studying Autographica californica nuclear polyhedrosis virus (AcMNPV) and other
baculovirus infections of cells of the cabbage looper moth, Trichoplusia ni (Fraser et al., 1996;
Fraser et al., 1995; Beames and Summers, 1990; Cary et al., 1989; Beames and Summers, 1988;
Fraser et al., 1985; Fraser et al., 1983). Investigators discovered two unique host-cell derived
2.4kb DNA sequences that had integrated into a specific region composed of two adjacent sites,
termed the few polyhedra (FP) locus of AcMNPV, wherein the four base-pair sequence “TTAA”
had been duplicated (Wang and Fraser, 1993; Beames and Summers, 1990; Cary et al., 1989;
Beames and Summers, 1988; Fraser et al., 1985; Fraser et al., 1983). The TTAA site was
determined to be a recognition site for piggyBac transposon insertion (Wang and Fraser, 1993).
Additionally, two 13-base pair (bp) ITRs, a set of two assymetrically-located 19bp internal
repeats (IRs), and another 7bp IR were detected in the piggyBac-transposed AcMNPV cells;
these features were eventually determined to be other key features of piggyBac transposon
integration (Carey et al., 1989; Fraser et al., 1985; Fraser et al., 1983).
PiggyBac transposons possess the ability to transfer very large (up to approximately 100kb)
insert regions and, have only shown minor losses of efficiency for inserts beyond 14kb (reviewed
in Vargas et al., 2018; reviewed in Zhao et al., 2016; Li et al., 2011; Wilson et al., 2007).
Previous uses of piggyBac transposons have included mouse germ line cells and HEK-293T cells
(reviewed in Vargas et al., 2018; Wilson et al., 2007; Ding et al., 2005). A significant advantage
of the piggyBac transposon system over other transposons, such as the extinct teleost fish-
derived, reconstructed/synthetic transposon system, Sleeping Beauty (SB), is the lack of any
“footprint” or base changes left behind after transposition (reviewed in Vargas et al., 2018;
reviewed in Zhao et al., 2016; Ivics et al., 1997; Radice et al., 1994). While SB is known to
leave behind a 3bp footprint in its insertion sites, piggyBac leaves its sites completely intact
(reviewed in Vargas et al., 2018; reviewed in Zhao et al., 2016).
44
One further advantage of the piggyBac system over SB and a few other transposon systems are a
reduced susceptibility to a phenomenon known as “overproduction inhibition” (OPI): a self-
regulatory feature of some transposases involving the decrease of transposase activity, suggested
to occur via transposase monomers forming dimers, preventing excision, that often occurs when
the copy number of a transposon and thus its transposase expression reach beyond a threshold
level (reviewed in Vargas et al., 2018; reviewed in Muñoz-López and García-Pérez, 2010;
Wilson et al., 2007). The piggyBac system has additionally been demonstrated to be modifiable,
with experiments demonstrating altered features such as: (a) increased excision and integration
efficiencies; (b) removed integration capabilities; and (c) drug inducibility (reviewed in Vargas
et al., 2018; Yusa et al., 2011; Li et al., 2013; Cadiñanos and Bradley, 2007). Despite the
significant advantages of the piggyBac transposon system, it nevertheless contains several
drawbacks, including a possible preference for integration in transcription start sites and areas of
transcription in target/host cells, as well as the potential for promoter disruption adjacent to the
integration area, due to minor transcriptional activity of the piggyBac 5’LTR (reviewed in
Vargas et al., 2018; Gogol-Döring et al., 2016; Cadiñanos and Bradley, 2007).
The Tol2 transposon, itself a member of the hobo/Ac/Tam3 (hAT) transposon family, and the
only naturally active vertebrate transposon, was originally isolated from the medaka fish
(reviewed in Zhao et al., 2016; Kawakami et al., 2000; Kawakami and Shima, 1999; Kawakami
et al., 1998). Tol2-affected fish were albino, due to the preference for Tol2 integration into a
tyronsinase gene in the medaka genome (Kawakami et al., 2000; Kawakami and Shima, 1999;
Kawakami et al., 1998). The original Tol2 element is 4.7kb in size and contains a four exon
transposase gene with flanking ITRs of varying length (reviewed in Muñoz-López and García-
Pérez, 2010; reviewed in Koga, 2004). The Tol2 transposon system has been successfully used
for gene transfer in a variety of frog (Xenpous laevis), zebrafish, threespine stickleback
(Gasterosteus aculeatus), and human cell types (reviewed in Kawakami, 2007; Howes et al.,
2017; Kikuta and Kawakami, 2009; Hamlet et al., 2006; Balciunas et al., 2006; Wu et al., 2006).
Unlike piggyBac, Tol2 is known to have no specific preference for integration and produces an
8bp duplication on integration (Grabundzija et al., 2010; reviewed in Muñoz-López and García-
Pérez, 2010; reviewed in Ivics et al., 2009; reviewed in Koga, 2004). Additionally, Tol2
excisions do leave behind one of two types of footprints, either the removal of the 5’ or 3’
terminal region, which has suggested the possibility of two distinct excision methods (reviewed
45
in Muñoz-López and García-Pérez, 2010; reviewed in Koga, 2004). Tol2 tranposons, much like
piggyBac transposons, have been demonstrated to similarly have a preference for integration
within transcriptional units and start sites (reviewed in Ivics and Izsvák, 2010; Grabundzija et al.,
2010). The cargo capacity of the Tol2 transposon is known to be at least 11kb, possibly higher,
however, this capacity is considerably dwarfed by the gargantuan 100kb capacity of the
piggyBac system (reviewed in Skipper et al., 2013; Balciunas et al., 2006; Urasaki et al., 2006).
Tol2’s transposition efficiency has been shown to be considerably lower than piggyBac’s, as
indicated by an experiment tracking the transposition frequencies of each in Henrietta Lacks
(HeLa) cells (Grabundzija et al., 2010). Tol2’s transposition efficiency at its peak activity was
only 1% in transposed HeLa cells, vs 3.5% for piggyBac (Grabundzija et al., 2010). Similarly,
the same study by Grabundzija et al. reported a higher OPI for Tol2 in HeLa cells (Grabundzija
et al., 2010). While the piggyBac transposon used by Grabundzija et al. required only 50ng of
“helper plasmid” (the transposase-encoding plasmid) to reach its peak transpositional activity,
Tol2 required as much as 125ng of its own helper plasmid (Grabundzija et al., 2010). Overall,
the work of Grabundzija et al. generally demonstrated the Tol2 transposon system to be less
efficient than the piggyBac system, in a variety of factors ((Grabundzija et al., 2010). An
additional study comparing Tol2 and piggyBac transposon efficiencies for gene transfer to
human primary T cells reached similar conclusions as Grabundzija et al. (Huang et al., 2010).
In summary, transposons have certainly shown promise in a variety of species and a plethora of
cell types as gene transfer vehicles (reviewed in Vargas et al., 2018; reviewed in Skipper et al.,
2013; reviewed in Ivics et al., 2009). Although they are limited by such drawbacks as OPI,
tendencies for integration within transcriptional units, and in the case of Tol2 and SB, excision
footprints, they nevertheless present themselves as largely more suitable gene transfer vectors in
comparison to viral vectors (e.g., lentivirus), both due to increased cargo capacity and reduced
biosafety among other factors (reviewed in Vargas et al., 2018; reviewed in Skipper et al., 2013;
reviewed in Ivics et al., 2009).
46
Chapter 2: Research Aims
Overarching Goals, Questions, & Technical Aims
As discussed in Chapter 1- Sections 2.1.2 & 3.1, my MSc project of creating transgenic,
targetable NPCs for selective prodrug ablation exists in the overarching context of the
unanswered questions in SCI neuroregeneration of:
(1) “Is (a) remyelination, or (b) neuronal regeneration (neurite outgrowth)/axonal and synaptic
plasticity reestablishment the more effective and efficient strategy for SCI neurorepair (and
eventually pursuing translational therapeutic treatments)?”
(2) “Is long-term integration of transplanted cells (e.g., establishment of synaptic connectivity
from transplanted NPC-derived neurons, or large-scale remyelination directly by transplanted
OPC-derived oligodendrocytes) necessary for providing significant physical repair and
functional recovery, or is the combination of the short-term mechanisms of: (i) trophic support
(e.g., secretion of positive growth factors – BDNF/GDNF etc.), and (ii) immune modulation
(e.g., downregulating production of Il-6, IL-1β, or TNF-α) sufficient?”
and (3) “How much proliferation of transplanted hiPSC-NPCs is necessary and for how long
must it occur to provide effective SCI physical repair and functional recovery?”
(reviewed in Ahuja et al., 2017a&b; Ahuja and Fehlings, 2016; Siddiqui et al., 2015; Badner et
al., 2017; Khazaei et al., 2017).
Cell ablation may be used to attempt to answer the above three questions, in addition to several
others in regards to NPC transplants in a neuroregeneration context. Future experiments beyond
the scope of my current work could potentially make use of my suicide-gene-expressing NPCs in
transplant studies into SCI-model rodents, with prodrug-mediated ablations and evaluations both
of the loss of physical, cellular and tissue repair in the SCI lesion, as well as the reductions in
functional recovery gained (if any) from non-ablated NPC-transplanted SCI rats over control
(sham and vehicle injured) rodents. Although such experiments are far beyond the scope of my
current MSc project, I nevertheless wish to frame my work in its larger picture for future
potential uses.
47
As my MSc project is a more methods focused approach, in lieu of traditional hypotheses, I have
developed a set of three key research aims to break down the fundamental aspects of my work.
The research aims I have developed towards creating selectively-targetable NPCs are:
1) Obtain an effective and efficient gene transfer vector (namely the piggyBac and Tol2
transposons for my NTR-NPC and HSV-TK NPCs respectively).
2) Generate Suicide-Gene expressing NPCs (an especially lengthy process with regards to the
NTR-construct NPCs, due to the two month-long culturing time for the monoclonal lines, and
only a very short (approximately 8-hour timeframe) for the puromycin purification of HSV-TK-
NPCs); and,
3) Perform drug directed ablations (accomplished using Mtz and GCV/BVDU for the NTR
and HSV-TK NPCs respectively).
48
Chapter 3: Materials and Methods
1) NTR Insert Subcloning: Lentivirus->PiggyBac
The initial steps undertaken in my MSc project involved the subcloning of the three promoter-
NTR inserts (CMV, DCX, and MBP) from a pre-existing lentiviral plasmid vector into a
piggyBac transposon vector (displayed in Figures 1a-1d in Chapter 4: Results). Template
DNA of each NTR-promoter construct was produced through plating and streaking to acquire
single colonies of DH5α E. coli from bacterial stabs previously made available, followed by
culturing the single colonies using antibiotic selection with kanamycin (50µg/mL), and finally
maxiprep plasmid purification (alkaline lysis-neutralization-silica column elution).
Bacterial stabs were streaked on 10cm culture plates (Sarstedt TC Dish, 100 Standard Catalogue
(Cat.) #83.3902) containing 15mL of solidified agar (1.2%; Difco Cat. #0140-01) with Lennox-
broth (LB; 20g per L of H2O; Sigma Aldrich Cat. #L3022) and grown overnight at 370C in a
Lab Companion IB-11E 150L Incubator (Jeio Tech Company) to acquire single colonies of
plasmid-bearing E. coli. Single colonies of each plasmid-containing bacteria were picked using
10µL micropipette tips and cultured in 2L Erlenmeyer flasks containing 500mL of LB media
(20g per L of H2O; Sigma Aldrich Cat. #L3022) overnight at thirty-seven degrees Celsius with
mild shaking (225rpm) in a floor-model MaxQ5000 bacterial culture incubator/shaker
(Thermofisher Scientific Cat. #SHKE5000). Purified plasmid DNA was acquired via alkaline-
lysis-neutralization, silica gel column adherence, elution and isopropanol precipitation with 70%
ethanol washes (GeneAid Maxiprep Kit Cat. #PM010; 1 maxiprep per 250mL of culture, 2
maxipreps per plasmid). Final plasmid DNA was resuspended in 1mL of sterile molecular grade
H2O (Wisent Multicell Cat. #809-115-CL) per maxiprep (250mL or one-half of each plasmid’s
culture) in 1.7mL graduated centrifuge tubes (Froggabio Cat. #LMCT1.7B).
For the first portion of NTR subcloning, I performed PCR amplification of each of the three
NTR-promoter constructs using three pairs of primers (ACGT Corporation) designed to not only
amplify the constructs, but also additionally extend them with flanking MluI restriction enzyme
recognition sites. The primers used for PCR amplification of the NTR constructs were as
follows:
49
1) CMV-NTR:
a) Forward (5’-> 3’) Primer: MluI-CMV-F:
ACGTACGCGTtccctatcagtgatagagatctccc
b) Reverse (3’-5’) Primer: MluI-CMV-R:
TCGAACGCGTactttccacaccTCTAGAAAAGTCG
2) DCX-NTR:
a) Forward (5’->3’) Primer: MluI-DCX-F:
ACGTACGCGTactccctatcagtgatagagatctccc
b) Reverse (3’-5’) Primer: MluI-CMV-R:
TGCAACGCGTcggagttaggggcgggac
2) MBP-NTR:
a) Forward (5’->3’) Primer: MluI-MBP-F:
ACGTACGCGTactccctatcagtgatagagatctccc
b) Reverse (3’-5’) Primer: MluI-MBP-R:
TGCAACGCGTactttccacaccTCTAGAAAACAATCCC
The MluI enzyme restriction digest site added was as follows (whereby | lines between bases
indicate the precise position of the asymmetric restriction digest cut):
5’ … A| C G C G T … 3’
3’ … T G C G C | A … 5’
The PCR amplification of NTR-promoter constructs was performed using a Mastercycler® pro
thermocycler (Eppendorf Cat. #950030010) using the following PCR programs (all identical):
50
1) CMV-NTR:
980 C - 2 minutes (Initiation)
35x Cycles of [section enclosed within bolded brackets]:
[ 980 C - 15 seconds (Denaturation)
650 C - 30 seconds (Annealing)
720 C - 1 minute + 10 seconds (Elongation) ]
720 C - 5 minutes (Final Elongation)
40 C - hold (Final Hold)
2) DCX-NTR:
980 C - 2 minutes (Initiation)
35x Cycles of [section enclosed within bolded brackets]:
[ 980 C - 15 seconds (Denaturation)
650 C - 30 seconds (Annealing)
720 C - 1 minute + 10 seconds (Elongation) ]
720 C - 5 minutes (Final Elongation)
40 C - hold (Final Hold)
3) MBP-NTR:
980 C - 2 minutes (Initiation)
35x Cycles of [section enclosed within bolded brackets]:
[ 980 C - 15 seconds (Denaturation)
51
650 C - 30 seconds (Annealing)
720 C - 1 minute + 10 seconds (Elongation) ]
720 C - 5 minutes (Final Elongation)
40 C - hold (Final Hold)
For each NTR-insert PCR amplification, 400µL master mixes were set up, then aliquoted into 16
x 25µL reactions. The PCR master mixes for all NTR constructs were created as follows:
a) Molecular Biology Grade, Sterile, Double-Distilled Water (DDH2O) (Wisent Multicell Cat.
#809-115-CL): 200µL (12.5µL per 25µL reaction)
b) 5x Q5® Reaction Buffer (New England Biolabs Cat. #B9027S): 80µL (5uL per 25µL
reaction)
c) 10mM Deoxynucleotide (dNTP) Solution Mix (New England Biolabs Cat. #N0447S): 8µL
(0.5µL per 25µL reaction)
d) Forward Primer (MluI-CMV/DCX/MBP-F): 4µL (0.25µL per 25µL reaction)
e) Reverse Primer (MluI-CMV/DCX/MBP-R): 4µL (0.25µL per 25µL reaction)
f) Template DNA (lentiviral CMV/DCX/MBP NTR- 1ng/µL): 16µL (1µL per 25µL reaction)
g) 5x Q5® High GC Enhancer (New England Biolabs Cat. #M0491S): 80µL (5µL per 25µL
reaction)
h) Q5® High-Fidelity DNA Polymerase (New England Biolabs Cat. #M0491S, 2,000 units/µL):
8µL (0.5µL per 25µL reaction)
PCR amplified NTR-promoter inserts (20µL volume of each 25µL reaction) were then run on
1% agarose (Invitrogen Ultrapure Agarose Cat. #16500-500) DNA gels with 20 lanes, containing
2.5µL of SYBRTM Safe DNA gel stain (Invitrogen Cat. #S33102) at 80V for 100 minutes in a
Wide Mini-Sub® Cell GT Horizontal Electrophoresis System with a 15 x 10 cm tray (Bio-Rad
Cat. #1704468) powered by a PowerPac™ HC High-Current Power Supply (Bio-Rad Cat.
52
#1645052). Finished gels were imaged using the ChemiDoc™ XRS+ Imaging System (Bio-Rad
Cat. #1708265) on a Universal Hood III gel dock (Bio-Rad Cat. #731BR02333) with exposure
times ranging from 0.5 seconds to 4 seconds.
Once the NTR-promoter insert fragments had been PCR amplified with added flanking MluI
sites, the subsequent steps in NTR subcloning entailed fragment isolation (from an agarose
electrophoresis DNA gel) of a large quantity (400µL of each PCR reaction mix) of each PCR-
amplified NTR insert, followed by subsequent steps of purification, resuspension, MluI-
digestion, and repeated purification+resuspension.
Each PCR-amplified, NTR insert was run on a 1% agarose preparatory gel (precise
materials/catalogue numbers as described directly above) at 60V for 100 minutes. Each
preparatory gel consisted of 4 large wells, each loaded with 2x (50µL PCR NTR-fragment
reaction mix + 10µL gel loading dye, purple (6x - New England Biolabs Cat. #B7024S), as well
as 3uL (150ng) of a diluted (50ng/µL) 1kb plus DNA ladder (ThermoFisher Scientific Cat. #
10787018).
PCR-amplified-NTR insert fragments were then isolated from each preparatory gel using fresh,
single edge Fisherbrand™ razor blades (ThermoFisher Scientific Cat. #12-640) by placing each
gel under UV illumination with a benchtop UV transilluminator. Purification of the NTR
fragments from the extracted agarose gel slabs was accomplished first through column
purification using homemade columns composed of Fisherbrand™ Premium Microcentrifuge
Tubes: 0.6mL (ThermoFisher Scientific Cat. #502GRDBFP) with extremely small holes poked
through the bottom centre of each tube with a flame-heated, 25-gauge needle (Sigma-Aldrich BD
Precisionglide® syringe needles Cat. #Z19406) mounted on a 1mL syringe (Sigma-Aldrich Cat.
# Z683531) and placed inside 2mL collection tubes (borrowed from a plasmid miniprep kit-
QIAGEN Cat. #27104) stuffed approximately 1/3 full with glass wool (Sigma-Aldrich Cat. #
18421). The NTR fragment agarose-gel isolated slabs were then loaded into 2 columns each
(based on agarose slab size/volume) and centrifuged at 8000g for 2 minutes.
The aqueous flow-throughs containing each of the NTR fragments were then purified via phenyl-
chloroform (Sigma-Aldrich Cat. #P1944) purification. NTR fragments were mixed with phenyl-
chloroform in a volume 1:1 ratio in 1.7mL graduated centrifuge tubes (Froggabio Cat.
#LMCT1.7B), mixed vigorously via shaking and inverting, then centrifuged at 14,000g in an
53
ultracentrifuge, then subsequently the top/clear layer of each fragment DNA (approximately
700µL) was pipetted out and transferred to a fresh 1.7mL centrifuge tube. Post-purified NTR
fragment DNA was then precipitated using 95% anhydrous ethanol (EtOH) (Sigma-Aldrich Cat.
#676829) added at a 2.5:1 ratio of EtoH:fragment DNA with the addition of 10% total final
volume worth of sodium acetate (Sigma-Aldrich S2889), and frozen at -200C for one hour. NTR
fragment DNA was then pelleted via centrifugation at 14,000g for 20 minutes, and the
supernatant of each tube was drained, followed by the addition of 100µL of 70% EtOH with
gentle mixing (tapping of tubes), then centrifuged again at 14,000g for 10 minutes. After the
final centrifugation step, the 70% EtOH supernatant was drained carefully, and the NTR
fragment DNA tubes were left to dry until the ethanol smell dissipated, then finally resuspended
in 28µL of molecular grade DDH2O. For each purified NTR fragment, two tubes of 28µL were
obtained and the tubes were combined into one tube of 56µL per fragment.
After resuspension, the purified DNA of each NTR fragment was then run on a 1% agarose DNA
electrophoresis gel for 100 minutes at 80V for visual inspection of the purified fragments. For
the DNA gel, 2µL of each purified NTR fragment DNA was mixed with 4µL of molecular grade
sterile DDH2O and 4µL of 6x gel loading dye; 3µL (150ng) of 1kb plus DNA ladder was
additionally included in the gel. MluI digestions of the PCR-amplified, purified NTR fragments
(amplified to contain flanking MluI restriction sites) were then undertaken in 100µL total
volume, using a mixture of 68µL of molecular grade sterile DDH2O, 20µL of each NTR
fragment, 10µL of 10x buffer 3.1 (New England Biolabs Cat. #B7203S), and 2µL of MluI
enzyme (New England Biolabs Cat. #R0198S). The NTR fragment MluI digestions were carried
out at 370C overnight (approximately 15 hours). Post-MluI digestion, the NTR fragments were
purified via phenyl chloroform purification and resuspended in 20 µL molecular grade DDH2O
each (identical methods as described above). 2µL of each of the purified and resuspended, MluI-
digested NTR fragments (with 4µL of molecular grade sterile DDH2O and 4µL of 6x gel loading
dye) was then run on a 1% agarose DNA gel at 80V for 100 minutes.
The piggyBac vector plasmid (into which the MluI-digested NTR fragments were to be cloned)
was subsequently then itself digested with MluI. A reaction mix consisting of a total volume of
100µL was created using 82.3µL of molecular grade sterile DDH2O, 10µL of buffer 3.1, 5.7µL
(approximately 2µg) of piggyBac plasmid DNA, and 2µL MluI enzyme. The piggyBac vector
plasmid MluI digestion was carried out at 370C for 4 hours.
54
Ligations of the MluI digested (and purified & resuspended) NTR fragments+piggyBac vector
were then set up using 23µL of molecular grade sterile DDH2O, 4µL of 10x T4 ligase buffer
(New England Biolabs Cat. #M0202S), 10µL of insert DNA (the MluI-digested/purified NTR
fragments), 1µL of vector DNA (the MluI-digested, linearized piggyBac plasmid vector), and
2µL of T4 DNA ligase (New England Biolabs Cat. #M0202S). The four 40µL ligation reactions
(3 for each NTR fragment insert plus 1 control ligation of just the MluI-digested piggyBac
vector) were each split into 2x20µL reactions in 200µL PCR tubes (Bio-Rad Cat. #TFI0201) and
run overnight (approximately 15 hours) at 160C in a Mastercycler® pro thermocycler (Eppendorf
Cat. #950030010).
The next morning, the NTR-piggyBac ligation reaction mixes were precipitated with 100%
EtOH and resuspended in 12µL of molecular grade sterile DDH2O (protocol as described above),
then used for electroporation to transform DH10β E. coli. Transformations were performed
using 35 µL of 1/5th diluted (in 10% glycerol) ElectroMaxTM DH10β E. coli cells (Invitrogen
Cat. #18290-015), with 5µL of each ligation reaction mix in an electroporator (Eppendorf
Multiporator Cat. # 940000602) using 2500V electroshocks for 5ms. 400µL of SOC media
(New England Biolabs Cat. #B9020S) was added to the electroporated bacterial cells, which
were then incubated at 370C for one hour. After the recovery incubation, the transformed
bacterial cells were pelleted gently at 500g for 5 minutes, and the volume of SOC media was
adjusted to 200µL, followed by gentle resuspension by pipetting. Transformed DH10β cells
were then plated on 10cm dishes with LB+agar (1.2%). For each ligation reaction’s transformed
DH10β cells, 50µL was used per each of 4 plates containing 50µg/mL of kanamycin. The cells
on each plate were distributed via shaking with glass plating beads (Novagen Cat. #71013-4),
then left overnight (approximately 15 hours) for a 300C incubation.
Single colony DH10β clones were picked from each NTR-fragment insert + piggyBac vector
ligation-reaction plate and cultured in 14mL plastic culture tubes (Corning Life Sciences Cat.
#C352006) with 6mL of LB media containing 50 µg/mL of kanamycin at 300C with 225rpm of
shaking in a benchtop shaking incubator (Corning Life Sciences Cat. #EF18354) for 15 hours.
The next day, DNA from the single-colony-derived clones for each NTR insert + piggyBac
vector was miniprepped using 2.1mL of each clone (QIAGEN Cat. #27104, protocol contained
therein). Each clone’s miniprep DNA was eluted using 35µL of molecular grade sterile DDH2O.
55
The miniprepped DNA from each NTR-piggyBac clone (14 for CMV-NTR-piggyBac, 5 for each
of DCX- and MBP-NTR-piggyBac) was then digested with MluI in order to check for the
presence of each insert fragment (as the MluI site was preserved during the ligation and now
flanked each NTR insert in the piggyBac vector (see Chapter 4: Results, Figure 1e-g). MluI
reaction mixes were setup using 10.5µL of molecular grade sterile DDH2O, 2µL of buffer 3.1,
7µL of each clone’s miniprep DNA, and 0.5µL of MluI enzyme, and digested at 370C for 1 hour.
The MluI-digested miniprep clone DNA (20 µL per digest + 5µL of purple 6x loading dye) was
then run on two 0.8% agarose gels with 20 wells each (due to the high number of clones) at 80V
for 100 minutes, then imaged (using the same gel imager described earlier in this chapter). For
CMV-NTR-piggyBac, clones #11-14 (termed C11-C14) were found to possess the CMV-NTR
insert (via release of a 2.1kb fragment after MluI digestion). For DCX-NTR-piggyBac and
MBP-NTR-piggyBac, clones #5 (D5) and #1-5 (M1-M5) respectively were found to possess the
DCX- and MB-NTR inserts (via release of the 3.5kb fragments after MluI digestions).
Additional amounts of each positive clone’s plasmid DNA was acquired via adding 5mL of LB
(with 50µg/mL kanamycin) to the remaining cultured clone stock in 14mL culture tubes,
growing overnight at 300C with shaking (225rpm), followed by plasmid miniprep (as described
earlier).
A series of follow up restriction digests (25µL each), using 5µL (of 35µL total) of miniprep
clone DNA, and 0.5µL of each restriction enzyme (listed below) were set up for the positive
clones C11-C14, D5, and M1-M5, using:
NdeI single digest (New England Biolabs Cat. #R0111S) (C11-C14, M1-M5, and D5);
SnaBI (New England Biolabs Cat. #R0130S) single digest (C11-C14);
SnaBI + Bstz17I (New England Biolabs Cat. #R3594S) double digest (C11-C14);
HindIII (New England Biolabs Cat. #R0104S) single digest (C11-C14);
EcoRV (New England Biolabs Cat. #R0195S) (M1-M5);
XhoI (New England Biolabs Cat. #R0146S) single digest (M1-M5);
XhoI + EcoRV double digest (M1-M5);
56
XhoI + NdeI (M1-M5, and D5);
Bstz17I single digest (M3);
NdeI + Bstz17I double digest (M3); and,
EcoRV + NdeI souble digest (D5).
The follow-up restriction digests for the positive clones were run on 0.8% agarose gels and
confirmed CMV-NTR-piggyBac clones- C13 and C14, DCX-NTR-piggyBac clones D5, and
MBP-NTR-piggyBac clone M3 to not only possess the appropriate respective NTR insert, but
also be the correct expected size of each plasmid restriction fragment, so as to rule out the
possibility of any major (~1kb+) deletions through hyper-recombination during DH10β
incubation after electroporation and plating single colony clones.
The final steps of subcloning of the NTR-promoter constructs from their original lentiviral
plasmid vectors, into the piggyBac transposon vector, entailed the use of BsaI (New England
Biolabs Cat. #R0535S) digests to remove an unrelated insert in the plasmid, which otherwise
could have potentially interfered with NTR expression or transposon insertion in host NPC
genomes. The unrelated insert was flanked by BsaI restriction sites, thus allowing simple single
BsaI digests followed by ligations to re-circularize each linearized remaining, NTR-inserted,
piggyBac vector plasmid. BsaI digests of 100µL total volume were performed on clones C13,
C14, M3, and D5 using: 50µL (500ng) of miniprepped clone DNA (multiple minipreps were
combined per clone to acquire enough plasmid DNA), 3µL of BsaI enzyme, 10µL of 10x
CutSmart® buffer (New England Biolabs Cat. #B7204S), and 37µL of molecular grade sterile
DDH2O. All BsaI digests were incubated at 370C for approximately 15 hours overnight,
however 5µL of each reaction mix was removed after 1.5 hours.
The next morning, the BsaI-digested NTR-piggyBac clone DNA samples were purified with
phenyl chloroform, precipitated with 95% EtOH, and resuspended in 15µL molecular grade
sterile DDH2O. Additionally, the 5µL samples of each clone’s BsaI digest were run on a 0.8%
agarose gel at 80V for 100 minutes for visual inspection of linearization and removal of the
unrelated insert. Ligations were then set up to re-circularize the NTR-piggyBac clone plasmids,
using 40µL total volumes (later split into 2x 20µL reactions) per BsaI-digested clone composed
57
of a reaction mix of 2 µL of clone DNA, 2µL of T4 DNA ligase, 4µL of 10x T4 buffer, and
32µL of molecular grade sterile DDH2O. As described previously, ligation reactions were
carried out at 160C overnight (approximately 15 hours) in a thermocycler.
After precipitation and resuspension, the next day, the BsaI digested plasmid DNA of clones
C13, C14, D5, and M3 was electroporated (2500V for 5ms) into 35µL (per clone BsaI digest) of
1/5th diluted (in 10% glycerol) ElectroMaxTM DH10β E. coli cells. The same protocol for post-
electroporation, plating, and culturing of transformed bacterial cells was used as previously, with
the one difference that instead of dividing the final remaining 200µL of cells into 4 kanamycin
plates, all 200µL was plated onto one plate (per each BsaI-digested NTR-piggyBac clone-
transformation). Several single colonies were obtained the next day from each plate, which were
then cultured and miniprepped (protocol as described previously), and the resultant plasmid
DNA was screened for positive clones via BsaI (to check for the absence of the unrelated insert)
and MluI digests (to check for the presence of each promoter-NTR insert) (protocols as described
for diagnostic restriction digest sets above).
Final plasmid DNA of the promoter-NTR-piggyBac clones with the unrelated insert removed
was acquired via plasmid maxiprep protocol using an endotoxin-free purification kit (FroggaBio
Cat. #PM010, protocol described therein). Plasmid DNA concentration was quantified with a
nanodrop spectrophotometer (ThermoFisher Scientific Cat. #ND-2000). Two DNA pellets were
obtained per each of the three NTR-piggyBac clones (500mL of LB media with 50µg/mL
kanamycin culture volume per clone, 250mL culture per pellet), each of which was eluted in
1mL of molecular grade sterile DDH2O. Additionally, glycerol stocks comprised of 1mL of each
clone’s LB culture mix and 1mL of 50% glycerol (Sigma-Aldrich 56-81-5) were created and
stored at -800C as backups. Sequencing was additionally performed on the NTR-piggyBac
clones for complete confirmation using several primer sets (ACGT corporation).
2) NTR-NPC Monoclonal Lines and Mtz Ablations
The second major step in my MSc project, as indicated earlier in aim 2 of Chapter 2: Research
Aims, entailed transforming a line of human iPSC (hiPSC)-derived NPCs (supplied by Dr.
Mohamad Khazaei in our laboratory) with each of the three promoter (CMV/DCX/MBP)-NTR-
58
piggyBac transposon plasmids separately. The goal of such a step was to create three
independent NTR-NPC lines (CMV/DCX/MBP) with the NTR-promoter constructs integrated
into host cell genomes. Due to the nature of piggyBac transposons, and time constraints, random
integration was pursued, although site-directed integration (preferably into a vital/cell-life-
dependent gene) as achievable through Gateway cloning® (ThermofisherScientific) may have
been preferable (Grabundzija et al., 2010). In order to minimize the variation in NTR gene
expression (and thus Mtz susceptibility), monoclonal lines (derived from single NTR-NPCs)
were created via FACS and extremely-low (single cell) density plating was undertaken.
For the transformation of NPCs with the NTR-piggyBac plasmids, electroporation was
accomplished via the use of an Amaxa NucleofectorTM 2b (Lonza Cat. #AAB-1001). For each of
the three NTR-piggyBac plasmids, 1.0x106 NPCs in 1mL of serum free 9 media (SF9-
containing: Gibco™ Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12 -
ThermoFisher Scientific Cat. # 11320033); 1% of a penicillin-streptomycin mixture (P/S, 10,000
units/mL; Thermofisher Scientific # 15140122); 10nM B27 supplement - vitamin A
(ThermoFisher Scientific Cat. #12587010); 10nM BFGF, 10nM EGF; and 10nM heparin sodium
salt (Sigma-Aldrich Cat. # H3149)) were loaded into a curved glass electroporation cuvette
(accessory of Amaxa NucleofectorTM 2b) with 1µg (5µL) of NTR-piggyBac plasmid DNA, and
100µL of Nucleofector® Solution. Program A33 on the electroporator was selected, and after
completion of the transformations, 500µL of pre-warmed (370C) SF9 media was added to each
cuvette, and the transformed, NTR-NPCs were then taken for FACS.
Through FACS, accomplished with a flow cytometer (Beckman-Coulter Cat. #FC500),
successfully NTR-transfected NPCs (1-3%) were selected based on either mCherry (red)
fluoresence (for the ubiquitous CMV-NTR-NPCs), or green fluorescent protein (GFP)
fluorescence (for DCX- and MBP-NTR NPCs, as all NTR-piggyBac transposon plasmids
possessed a GFP marker for visual identification of successful transformation) and single cells of
each of the three types were plated with SF9 media in multiple wells of one 96 well plate
(Sarstedt Cat. #83.3924.005) per each of the three promoter-NTR combinations. Monoclonal-
derived cell groupings (referring to each plated well) exhibiting the brightest mCherry (CMV-
NTR-NPCs) or GFP (DCX- and MBP-NTR NPCs) were selected and continually cultured and
expanded (and gradually transferred to larger volume cell culture plates) over a two-month
timeframe to eventually produce monoclonal, NTR-NPC lines consisting of approximately 1.5-
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2.0x107 cells each per line. Ten backups of approximately 1.0x106 cells each per NTR-NPC
monoclonal line were created and placed in liquid nitrogen for long-term storage.
For ablation experiments with Mtz (Santa Cruz Biotechnology Cat. #CAS 443-48-1), 1.25x104
CMV-NTR-NPCs were plated in each well of a 24 well plate (Sarstedt Cat. #83.3922.005) in
SF9 media (500µL per well) using 15mm thickness, round glass cover slips (Bellco Glass Cat.
#1203J82) coated with Matrigel™ (Corning™ Matrigel™ Membrane Matrix Cat. #CB-40234A).
Twenty-four hours later, the media of each well was changed and switched to: SF9 media with
no Mtz (top row/row 1- wells 1-6, as a negative control); SF9 media with 2.5mM dissolved Mtz
(row 2- wells 7-12); SF9 media with 5mM dissolved Mtz (row 3- wells 13-18); and SF9 media
with 10mM dissolved Mtz (row 4- wells 19-24). DCX- and MBP- NTR NPCs (which would not
be capable of expressing NTR until differentiation into neurons and oligodendrocytes
respectively) were also plated at the same density in one 24-well plate per each cell line using the
same protocol as per CMV-NTR NPCs, however with the minor difference of using DMEM/F-
12 media containing 1% fetal bovine serum (FBS) (Sigma-Aldrich Cat. #F1051) and 1% P/S,
instead of SF9 media. DCX- and MBP-NTR NPCs were left in the serum-containing F-12 media
for approximately two weeks in order to induce differentiation (into a mixture of neurons,
oligodendrocytes, and astrocytes).
After each of a set of six timepoints (24-well plate vertical “columns”: column 1- 4 hours,
column 2- 12 hours, column 3- 24 hours, column 4- 48 hours, column 5- 72 hours, and column 6-
96 hours), the cover slips for CMV-NTR-NPCs for a corresponding vertical column (4 wells
each) were removed, transferred to the corresponding vertical column and well of a fresh 24 well
plate. The transferred cover-slip cells were then fixed with 1mL of 4.0% of paraformaldehyde
(PFA, Sigma-Aldrish Cat. #P6148), followed by 3x5 minute washes in 1mL of 1x (diluted 1:10
from 10x) phosphate-buffered saline (PBS, Wisent Bioproducts Cat. #311-012-LL), and finally
left in 1mL of 1x PBS. The same process described above for CMV-NTR NPCs (plating, Mtz
treatments, fixation, and qualitative visual observation) was additionally performed with a set of
WT-NPCs (hiPSC-derived NPCs with no NTR transgene) (supplied by Dr. Mohamad Khazaei
in our laboratory). Imaging of cells was performed once daily for 72 hours using an EVOS
FLoid™ Cell Imaging Station (ThermoFisher Scientific Cat. #4471136). Due to the lack of any
obvious visually-identifiable changes in cell density (and thus Mtz-mediated ablation) for any of
the Mtz concentrations or timepoints relative to each timepoint’s corresponding control well,
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further quantitative analysis (e.g., 4′,6-diamidino-2-phenylindole (DAPI) staining and
cytometry) was not pursued. A second attempt of the previously described Mtz ablation
experiment with CMV-NTR-NPCs was performed using an alternative source of Mtz (Sigma-
Aldrich Cat. #M3761).
3) NTR-Mtz Ablation Troubleshooting
Due to the unforeseen lack of ablational capability observed for any of the Mtz concentrations
and timepoints for the CMV-NTR-NPC line, two main avenues were pursed in order to attempt
to troubleshoot the inconclusive results observed (addressed further in Chapter 4: Results,
Figures 4-5). The first of these troubleshooting attempts entailed the evaluation of NTR enzyme
protein expression, while the second pursued the possibility of using a version of the NTR/NfsB
gene that had been optimized for human codon-preference usage (containing 144 silent
mutations) (Grohman et al., 2009).
There was unfortunately no readily available, commercial antibody for NTR, and thus a
comparison of the total protein extract of CMV-NTR-NPCs and WT-NPCs was made via
sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by staining
with Coomassie Brilliant Blue G-250 Dye (ThermoFisher Scientific Cat. #20279). The protein
extract of both CMV-NTR-NPCs and WT-NPCs was acquired via scraping cells (using
Corning® cell scrapers, Sigma-Aldrich Cat. #CLS3010) off 8 individual wells per each of the
two cell types, combining their media into one 15mL polypropylene conical centrifuge tube
(FroggaBio Cat. #TB15-500) per cell type, centrifuging at 290Gs for 5 minutes to pellet cells in
an Eppendorf 5810R 5 Amp version centrifuge with an A-4-81, 4x250mL swing bucket rotor
(Eppendorf Cat. #022627023) followed by removal of media supernatants, transferring to 1.7mL
ultracentrifuge tubes, resuspending first in 300µL of 1xPBS (with an extra identical
centrifugation step in between), then in a 300µL (per each cell type) lysis solution composed of
294µL radioimmunoprecipitation assay (RIPA) buffer (Sigma Aldrich Cat. # R0278), 3µL of
ethylenediaminetetraacetic acid (EDTA) (Sigma Aldrich Cat. #E6758), and 3µL of protease
inhibitor cocktail (Sigma-Aldrich Cat. #P8340 SIGMA), and finally via sonication.
The lysis-buffer-resuspended CMV-NTR- and WT-NPCs were placed on ice, and 3x10 second
cycles of sonication with a setting of 25W and 50% amplitude (with 45 second recovery periods
on ice in between in order to avoid overheating and denaturing proteins) was performed with a
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Vibra-Cell™ model VC50 AT sonicator (Sonics & Materials Inc., Dannbury, CT- discontinued
model) in a Fisherbrand™ Sound Enclosure (ThermoFisher Scientific Cat. #432B2). After
sonication, the lysis buffer CMV-NTR- and WT-NPC samples were centrifuged at 18,630g and
40C in a Scilogex refrigerated microcentrifuge D3024R (Scilogex Cat. #922015139999), and the
protein-extract-containing supernatant of each sample was aliquoted into 6x50µL aliquots in
1.7mL microcentrifuge tubes, then stored at -800C.
Prior to running SDS-PAGE and staining of the total protein of the CMV-NTR-NPC and WT-
NPC protein extracts, protein extract concentrations needed to be determined. Protein extract
concentrations were evaluated via performing a protein assay using spectrophotometry to
measure absorbances at 562nm with both the protein extract samples and protein samples of
known concentration, in order to establish a standard curve (using the Micro BCATM Protein
Assay Kit - ThermofisherScientific Cat. #23235, protocol described in detail within). For SDS-
PAGE, 20µg samples of each NPC protein extract was run on a gel composed of a 4%
acrylamide stacking gel affixed ontop of a 10% acrylamide resolving/separating gel in a
CriterionTM Cell (Bio-Rad Cat. #1656001) electrophoresis system for 30 minutes at 80V (for
protein samples to clear the stacking gel), followed by one hour and 30 minutes at 100V (for
protein samples to resolve).
Final staining of the total protein of each NPC extract on the gel was accomplished via 3x5
minute washes in DDH2O, followed by 30 minutes in Coomassie Brilliant Blue G-250 Dye, and
finally an overnight (approximately 15 hours) destaining wash in DDH2O. All washing, staining,
and destaining steps were performed with gentle rocking agitation on a Bio-Rad Mini Rocker
(BioRad Cat. # 1660710EDU). After staining, the gel was imaged using the ChemiDoc™ XRS+
System (Bio-Rad Cat. #1708265) on a Universal Hood III gel dock (Bio-Rad #731BR02333)
with exposure times ranging from 30 to 180 seconds.
The second portion of troubleshooting the NTR-Mtz ablation system for human iPS-derived
NPCs, as mentioned previously, entailed the use of a mutated variant of the NfsB/NTR gene that
had been optimized for human codon preference usage with 144 silent mutations (Grohman et
al., 2009). A construct composed of the human-codon optimized NfsB/NTR gene (HCO-NTR)
driven by the ubiquitous CMV promoter was cloned into the CMV-NTR-piggyBac plasmid
vector. Unlike the previous CMV-NTR construct used to generate the monoclonal line in
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Chapter 3: Section 2, the HCO-NTR construct did not possess a fluorescent marker tag/fusion-
protein gene, yet still possessed a GFP marker to identify transformed cells. Human-codon-
optimized-NPCs expressing NTR under the ubiquitous CMV promoter (HCO-NTR-NPCs) were
created using electroporation (as described previously) with 1µg of HCO-CMV-NTR plasmid
DNA, and successfully transformed cells were purified via antibiotic selection with puromycin
(Sigma-Aldrich #P8833) treatments of 1µg/mL for 5 hours, followed by 3 washes of 10 minutes
each using regular DMEM/F-12 media (with 10% FBS and 1% P/S).
For ablation experiments of HCO-NTR-NPCs, 1x106 cells were plated in 10cm, Matrigel™-
coated culture plates in SF9 media. Twenty-four hours later, the media of plates was replaced
with SF9 media mixed with pre-dissolved, pharmaceutical-grade Mtz to a final concentration of
6mM, from plastic intravenous sacks (IV Solution, Metronidazole, Metro-IV, 500mg, PAB®,
100mL, B.Braun Cat. # D5353-5224). The same procedure was repeated using non-
transformed/WT-NPCs. The HCO-NTR-NPC plates were then imaged daily for two days with
both brightfield and green-fluorescent channels using an EVOS FLoid™ Cell Imaging Station
(ThermoFisher Scientific Cat. #4471136).
4) HSV-TK Plasmid DNA Purification and Confirmation
HSV-TK plasmids used were obtained from Addgene (http://www.addgene.org), including the
Tol2 transposon plasmid, pKTol2P-PTK (Addgene #85599) containing a gene for a fusion
protein of HSV1-TK and puromycin resistance (puDeltatk) under the control of the ubiquitous
PGK promoter as well as the Tol2 transposase containing plasmid, pCMV-Tol2 (Addgene
#31823) (Clark et al., 2007; Balciunas et al., 2006; Chen and Bradley, 2000). Bacterial stabs
were streaked on 10cm culture plates (Sarstedt TC Dish, 100 Standard #83.3902) containing
15mL of solidified agar (1.2%; Difco #0140-01) with LB (20g per L of H2O; Sigma Aldrich Cat.
#L3022) and grown overnight at 370C in a Lab Companion IB-11E 150L Incubator (Jeio Tech
Company) to acquire single colonies of plasmid-bearing E. coli. Single colonies of each plasmid-
containing bacteria were picked using 10µL micropipette tips and cultured in 2L Erlenmeyer
flasks containing 500mL of LB media (20g per L of H2O; Sigma Aldrich L3022) overnight at
370C with mild shaking (225rpm) in a floor-model MaxQ5000 bacterial culture incubator/shaker
(Thermofisher Scientific #SHKE5000). Purified plasmid DNA was acquired via alkaline-lysis-
neutralization, silica gel column adherence, elution, and isopropanol precipitation with 70%
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ethanol washes (GeneAid Maxiprep Kit PM010; 4x maxi-preparations, 1 maxiprep per 250mL of
culture, 2 maxipreps per plasmid). Final plasmid DNA was resuspended in 1mL of sterile
molecular grade H2O (Wisent Multicell 809-115-CL) per maxiprep (250mL or one-half of each
plasmid’s culture) in 1.7mL graduated centrifuge tubes (Froggabio LMCT1.7B).
Restriction digests of plasmid pCMV-Tol2 were performed with 200ng of plasmid DNA using
the enzyme combinations of 1.) BsaI (New England Biolabs #R0535S) and SnaBI (New England
Biolabs #R0130S); and 2.) BsaI (New England Biolabs #R0535S) and BglII (ThermoFisher
Scientific # ER0081). Similarly, restriction digests of plasmid pKTol2P-PTK were performed
with 200ng of plasmid DNA using the enzyme combinations of 1.) NheI (New England Biolabs
#R0131S) and SnaBI (New England Biolabs #R0130S); and 2.) BsaI (New England Biolabs
#R0535S) and BglII (ThermoFisher Scientific # ER0081). For all restriction digests, enzyme-
DNA-buffer mixtures were incubated for one hour at thirty-seven degrees Celsius in a 150L
incubator (Jeio Tech Company Lab Companion IB-11E).
Subsequently, the restriction digests of each plasmid were run on a 1% agarose (Invitrogen
Ultrapure Agarose #16500-500) gel with 20 lanes at 80V for 100 minutes in a Wide Mini-Sub®
Cell GT Horizontal Electrophoresis System with a 15 x 10 cm tray (Bio-Rad #1704468) powered
by a PowerPac™ HC High-Current Power Supply (Bio-Rad #1645052). Finished gels were
imaged using the ChemiDoc™ MP Imaging System (Bio-Rad) on a Universal Hood III gel dock
(Bio-Rad #731BR02333) with exposure times ranging from 0.5 seconds to 4 seconds. Similarly,
to the NTR-piggyBac plasmids created through subcloning in Chapter 3: Section 2, both
plasmids CMV-Tol2 and KTol2P-PTK were further confirmed via sequencing (primers supplied
by ACGT Corporation).
5) Mixed NTR and HSV-TK NPC GCV Ablation
The initial usage of the HSV-TK+GCV ablation system in my MSc project attempted to assess
bystander effects through applying GCV to a mixed population of HSV-TK+ NPCs and non-
HSV-TK-expressing NPCs. For the HSV-TK- NPCS, HCO-NTR-NPCs (from Chapter 3:
Section 3) were selected due to their immediate availability. The same line of hiPSC-derived
NPCs was used to generate HSV-TK+ NPCs, and the transformations with both plasmids CMV-
Tol2 (Tol2 transposase) and KTol2P-PTK (Tol-2 transposon carrying a puromycin-
resistance+HSV-TK fusion protein gene) were carried out identically as described for the three
64
promoter NTR-NPC lines (Chapter 3, Section 2) and HCO-NTR-NPCs (Chapter 3, Section 3).
500ng of each plasmid of the two-plasmid, Tol2 system was used during the transformation,
rather than 1µg for the single-plasmid systems described earlier (Chapter 3, Sections 2 & 3).
For the GCV bystander evaluations, 1.0x106 cells, of either: HCO-NTR-NPCs (GFP+); HSV-TK-
NPCs; or a 1:1 mixture of both cell types, were plated in one 10cm plastic culture plate (Sarstedt
TC Dish, 100 Standard Cat. #83.3902) each (per cell type used) in 10mL of SF9 media. One
additional plate of mixed HSV-TK and HCO-NTR NPCs was also set up as a negative control.
Twenty-four hours later, the media of each plate was changed and replaced with either: 1) three
experimental plates: SF9 containing 1µg/mL of dissolved GCV (powder dissolved in alkaline
DDH2O as described within InvivoGen protocol; InvivoGen Cat. # sud-gcv); or 2) negative
control mixed culture plate: regular SF9 media. All four plates were imaged once daily with
brightfield and green fluorescent channels of an EVOS FLoid™ Cell Imaging Station for a
period of 96 hours. One media change for all plates occurred at the 72-hour timepoint.
6) HSV-TK-NPC GCV and BVDU Bystander Ablations
In order to reduce bystander effects for future targeted cell killing studies beyond the scope of
this project, BVDU was selected as an additional ablation prodrug in combination with HSV-
TK-NPCs (reviewed in Dachs et al., 2009; Degrève et al., 1999). A mixed culture of HSV-TK-
NPCs and GFP+-WT-NPCs (a separate line provided by Dr. Mohamad Khazaei in our
laboratory) in a 1:1 ratio was set up wherein 5.0x104 cells (half HSV-TK-NPCs, and half GFP+-
WT-NPCs) were plated in each well (pre-coated with MatrigelTM) of two, six-well culture plates
(Sarstedt TC Plate 6 Well,Standard,F, Cat. #83.3920.005). After 24 hours, the wells of each plate
received the following 6 concentrations of GCV or BVDU respectively into one well each:
0µg/mL (negative control well); 0.0625µg/mL; 0.125µg/mL; 0.25µg/mL; 0.50µg/mL; and
1.0µg/mL. For the next four days, the media from each well of each plate was replaced with
fresh media containing the appropriate concentration of GCV/BVDU (Sigma-Aldrich Cat.
#B9647). All wells were imaged once daily with brightfield and green fluorescent channels of
an EVOS FLoid™ Cell Imaging Station for a period of 96 hours.
During the daily media changes with SF9 (containing the appropriate concentration of
GCV/BVDU), the old media of each well was collected in 15mL polypropylene conical
centrifuge tubes (FroggaBio #TB15-500). The 15mL tubes of collected media containing
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floating dead cells were centrifuged at 290Gs for 5 minutes to pellet cells in an Eppendorf 5810R
5 Amp version centrifuge with an A-4-81, 4x250mL swing bucket rotor (Eppendorf
#022627023). After centrifugation, the tubes were drained of media manually by gently pouring
into a 250mL waste beaker, then left inverted on a clean bench surface with paper towel for 2
minutes.
The cell pellets were then resuspended in 100µL of BD Cytofix/Cytoperm™
fixation/permeabilization solution containing 4.2% formaldehyde (BD Biosciences #554714)
using repeated pipetting to fully resuspend pelleted cells, then left for 20 minutes of fixation.
After fixation, the 15mL tubes of resuspended dead cells were centrifuged again at 290Gs for 5
minutes in the same Eppendorf 5810R swing-bucket rotor centrifuge as stated previously. The
supernatant of fixation solution was again drained manually, and the tubes were left inverted for
2 minutes to remove the remaining fixation solution. The fixed cell pellets were each finally
resuspended in 100µL of 1x PBS (Wisent Multicell #311-012-CL) with repeated pipetting, then
transferred to 1.7mL graduated centrifuge tubes (Froggabio LMCT1.7B), then stored at 40C.
Due to time constraints on my MSc project, as well as the apparent death of essentially all GFP+-
WT-NPCs after only twenty-four hours (as examined qualitatively in n=2 replicates), FACS and
cytometry of the fixed cells collected from the four timepoints of the two drug treatments was
not pursued.
7) HSV-TK-NPC GCV and BVDU Quantitative Ablations
The final step in the ablational studies carried out within this MSc project was the simple
quantitative evaluation of the ablational capabilities of GCV and BVDU against the line of HSV-
TK-NPCs (described earlier in Chapter 3: Sections 4-6). For both GCV and BVDU, four six
well culture plates (Standard Cat. #83.3920.005) were set up wherein 5.0x104 HSV-TK-NPCs
were plated in 2mL of SF9 media in each well (pre-coated with MatrigelTM). After 24 hours, the
media of all plates (8 total, 4 per prodrug representing one day treatment each) was changed, and
the wells of each plate received the following 6 concentrations of GCV or BVDU respectively
into one well each: 0µg/mL (negative control well); 0.0625µg/mL; 0.125µg/mL; 0.25µg/mL;
0.50µg/mL; and 1.0µg/mL (all dissolved in 2mL SF9 media).
Once daily for a period of 96 hours, one plate of HSV-TK-NPCs of both prodrug treatments (2
plates per prodrug/day) was removed from 370C incubation and the cells were fixed with 2mL of
66
4.0% of PFA (Sigma-Aldrich Cat. #P6148), followed by 2x5 minute washes in 1mL of 1xPBS
(Wisent Bioproducts Cat. #311-012-LL), and finally left in 1mL of 1x PBS. All wells were then
stained with 1-2µg/mL of DAPI for 10-30 minutes (later optimization required longer staining
times for improved fluorescence, image quality, and cytometry). All wells were imaged with the
brightfield channel of an EVOS FLoid™ Cell Imaging Station before fixation, as well as with
the brightfield and blue fluorescent channels post-fixation. A minimum of five representative
images were acquired per well. Estimation of the total remaining attached cells/well was
accomplished through “peak” analysis, whereby blue fluorescent, DAPI-stained nuclei (“peaks”)
were counted in ImageJ photo-analysis software (NIH). The mean number of total attached
cells/well was determined through extrapolating the mean number of cells in the surface area
(SA) covered by a representative image (approximately 0.37mm2) to the total SA per well
(approximately 1134mm2), a factor of ~3,057 times the SA of a representative image. The same
procedure was repeated for the 48, 72, and 96-hour timepoints, and n=3 replicates were
performed for the entire experiment.
As both the GCV and BVDU prodrugs used during the previous ablation experiments had been
diluted in dimethyl-sulfoxide (DMSO), up to a maximum final concentration of 0.4% DMSO in
culture media (the highest concentration of DMSO used for the 1.0µg/mL GCV/BVDU
concentrations), I believed it to be important to rule out the possibility of DMSO-induced cell
death, a minor possibility as indicated by previous literature (Yuan et al., 2014). In order to fully
determine if DMSO, and not prodrug treatment could be culpable for inducing NPC death, I
plated 5.0x104 NPCs in two wells of a six well culture plate on MatrigelTM with 2mL of SF9
media. Twenty-four hours later, the media of both wells was changed and replaced with either a)
SF9 containing 0.4% DMSO (the highest DMSO concentration used for prodrug dilution), or b)
0.4% PBS then returned to 370C incubation. After 48 hours, both wells were imaged with the
brightfield channel of an EVOS FLoid™ Cell Imaging Station and qualitatively evaluated for
obvious changes in cell density.
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Chapter 4: Results
1) NTR-PiggyBac Subcloning
The earliest portion of my MSc project entailed the subcloning of three promoter- (CMV, DCX,
and MBP) NTR inserts (containing an NTR-mcherry fusion protein) from a lentiviral plasmid
vector (Figures 1a-1c) into a piggyBac transposon vector (Figure 1d). After several
unsuccessful attempts at removing the NTR inserts via restriction digests at flanking regions,
blunting both the insert and vector ends via Klenow (E. coli DNA polymerase I) fragment action
(not shown), eventual success in obtaining positive clones was achieved through adding flanking
MluI restriction sites via PCR amplification of the NTR inserts. Both the NTR inserts and
piggyBac vector plasmid were then digested with the MluI restriction endonuclease and ligated
with T4 DNA ligase (described in depth in Chapter 3: Materials and Methods). DH10β E.
coli were transformed through electroporation and grown at 300C on 10cm agar plates to obtain
single colonies. Plasmid DNA from cultured single colony clones was then extracted via
miniprep protocol and positive clones containing the successfully ligated piggyBac vector
plasmid with promoter-NTR inserts were confirmed both by MluI restriction digests and
sequencing (Figure 2a-d).
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Figure 1a: CMV-NTR: original lentiviral plasmid ~10.9kb
(top) and subcloning insert form ~2.1kb (bottom). The
original subcloning attempt involved cutting the CMV-NTR
insert out of the lentiviral plasmid via an NheI+XbaI double
restriction digest with blunt end cloning. The successful attempt
instead entailed the use of Q5-PCR amplification to add MluI
sites (near the XbaI (5113) and NheI (3062) sites respectively)
for “sticky-end” (5’ and 3’ overhangs) subcloning into the
piggyBac vector (see Figure 1d).
1a
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Figure 1b: DCX-NTR: original lentiviral plasmid ~12.3kb
(top) and subcloning insert form ~3.5kb (bottom). The
original subcloning attempt involved cutting the DCX-NTR
insert out of the lentiviral plasmid via an NheI+XbaI double
restriction digest with blunt end cloning. The successful attempt
instead entailed the use of Q5-PCR amplification to add MluI
sites (near the XbaI (6531) and NheI (3062) sites respectively)
for “sticky-end” (5’ and 3’ overhangs) subcloning into the
piggyBac vector (see Figure 1d).
1b
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Figure 1c: MBP-NTR: original lentiviral plasmid ~12.3kb
(top) and subcloning insert form ~3.5kb (bottom). The
original subcloning attempt involved cutting the MBP-NTR
insert out of the lentiviral plasmid via an NheI+XbaI double
restriction digest with blunt end cloning. The successful attempt
instead entailed the use of Q5-PCR amplification to add MluI
sites (near the XbaI (6526) and NheI (3062) sites respectively)
for “sticky-end” (5’ and 3’ overhangs) subcloning into the
piggyBac vector (see Figure 1d).
1c
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Figure 1d: PiggyBac Transposon Vector Plasmid. All three
promoter-NTR inserts (Figures 1a-c) were subcloned into the
above vector at the MluI restriction site (7336, outlined in red)
via PCR amplification to add flanking MluI restriction sites to
each NTR insert, followed by the MluI digest of the vector and
all inserts to create compatible 5’ and 3’ overhang “sticky-ends”.
An unrelated 600bp insert (5335 -> 6022, outlined in blue) was
removed following ligation of the NTR inserts from each of the
three resultant NTR-piggyBac plasmids via BsaI restriction
digests (due to sites flanking the insert) and self-ligation.
1d
72
1e
Figure 1e: PiggyBac Transposon Vector containing the
subcloned CMV-NTR insert. The primers used to PCR-amplify
the CMV-NTR insert and add flanking MluI restriction sites (8768
and 6653) are indicated in purple (MluI-CMV-F (6653-6683) and
MluI-CMV-R (8743-8774)). Positive clones were confirmed both
through MluI restriction digest to release the CMV-NTR insert (see
Figure 2c) and through sequencing of a region spanning 500bp both
up and downstream of the CMV-NTR insert (indicated in blue
above).
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Figure 1f: PiggyBac Transposon Vector containing the subcloned
DCX-NTR insert. The primers used to PCR-amplify the DCX-NTR
insert and add flanking MluI restriction sites (10,340 and 6653) are
indicated in purple (MluI-DCX-F (6653-6685) and MluI-DCX-R
(10,322-10,346)). Positive clones were confirmed both through MluI
restriction digest to release the DCX-NTR insert (~3.5kb, see Figure 2d)
and through sequencing of a region spanning 500bp both up and
downstream of the DCX-NTR insert (indicated in blue above).
1f
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Figure 1g: PiggyBac Transposon Vector containing the subcloned
MBP-NTR insert. The primers used to PCR-amplify the MBP-NTR
insert and add flanking MluI restriction sites (10,175 and 6645) are
indicated in purple (MluI-MBP-F (6645-6677) and MluI-MBP-R
(10,147-10,181)). Positive clones were confirmed both through MluI
restriction digest to release the MBP-NTR insert (~3.5kb, see Figure 2b)
and through sequencing of a region spanning 500bp both up and
downstream of the MBP-NTR insert (indicated in blue above).
1g
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2c 2d
Figure 2: Agarose DNA Gel Restriction Digests of NTR Inserts and PiggyBac Transposon Vector. a)
PCR amplified and MluI-digested: 1=CMV-NTR insert; 2&3=DCX&MBP-NTR inserts; 4=piggyBac vector;
5=1kb+ DNA ladder. b) Successfully ligated MBP-NTR-piggyBac positive clone confirmations via MluI
digest and release of ~3.5kb MBP-NTR insert: 2,4,6,8,10=uncut MBP-NTR-piggyBac plasmid;
3,57,9,11=MluI-cut MBP-NTR-piggyBac; 12->13=uncut & MluI cut piggyBac vector respectively;
1&15=1kb+ DNA ladder. c) Successfully ligated CMV-NTR-piggyBac positive clone confirmations via
MluI digest and release of ~2.1kb CMV-NTR insert: 1,3,5,7,9,11,15=uncut CMV-NTR-piggyBac plasmid;
2,4,6,8,10,12,16=MluI cut CMV-NTR-piggyBac; 13->14=uncut & MluI cut negative clone (no CMV-NTR
insert); 17-18=uncut & MluI cut piggyBac vector; 19=1kb+ DNA ladder. d) Successfully ligated DCX-NTR-
piggyBac positive clone confirmations via MluI digest and release of ~3.5kb DCX-NTR insert: 1=MluI cut
DCX-NTR-piggyBac plasmid; 2= uncut DCX-NTR-piggyBac; 3= 1kb+ DNA ladder.
2a 2b
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2) Mtz Treatment of CMV-NTR NPCs
After successful subcloning and confirmation of positive NTR-piggyBac plasmid clones (as well
as separate removals of the unrelated 600bp insert followed by self-ligation (not shown for
brevity)), endotoxin-free purified plasmid DNA was obtained via culturing a large volume
(500mL) of each plasmid’s single colony clone and extracting plasmid DNA through maxiprep
protocol. Three separate monoclonal lines of human iPSC-derived NPCs were generated via
electroporation with each of the NTR-piggyBac plasmids separately, FACS sorting, single cell
selection and prolonged expansion. CMV-NTR-NPCs would theoretically have been already
expressing NTR, and thus preliminary ablations using four concentrations of Mtz and six
timepoints were set up and evaluated qualitatively (Figure 3).
Figure 3: Preliminary attempts at Mtz ablation of CMV-NTR NPCs (e-h) in comparison with
WT-NPCs as a negative control (a-d). 1.25x104 NPCs were originally plated in each well of
separate 24-well plates, and after 24 hours, Mtz treatments began via media changes. Left columns
(a, c; & e, g ) depict the 4-hour timepount post-Mtz treatment, while the right columns (b, d; & f, h)
depict the 72 hour timepoints. Top rows (a-b; & e-f) indicate control wells receiving no Mtz
treatment, while the bottom rows (c-d; & g-h) received 10mM Mtz. For both WT-NPC controls and
CMV-NTR NPCs, cell proliferation and general cell quantities were relatively unchanged with
10mM Mtz (bottom rows) in comparison to control NPCs receiving no Mtz treatment (top rows).
Scale bars = 100µm.
3
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Mtz treatment up to 10mM did not appear to have any noticeably significant effects on total
remaining cell density of CMV-NTR NPCs after 72 hours. Due to such an unexpected result,
two principal attempts at troubleshooting these difficulties with NTR-Mtz ablation of my hiPSC-
derived, CMV-NTR-NPC line were pursued.
3) NTR-Mtz Ablation System Troubleshooting
Both (I) total protein staining (Figure 4- for investigating NTR translation, due to the lack of an
existing antibody), and (II) the generation of human codon-preference usage-optimized CMV-
NTR-NPCs (HCO-NTR-NPCs) (Figure 5), wherein the potential issue of low NTR transcription
due to prokaryotic (E. coli) codon-optimization could potentially be tackled were pursued as
options for troubleshooting NTR-Mtz -CMV-NTR-NPC ablation.
Figure 4: SDS PAGE and Coomassie Brilliant Blue G-250 Dye total protein stain of 20µg of protein
extract obtained from CMV-NTR-NPCs and WT-NPCs respectively by sonication. The polyacrylamide
gel was composed of a 4% stacking gel and a 10% resolving/separating gel, and was run for 30 minutes at
80V, followed by 1 hour and 30 minutes at 100V. A faint single band appeared (outlined in red) at the
approximate molecular mass (~50kDA) of the NTR-mcherry fusion protein for the CMV-NTR protein
extract, compared to the less clear band smear evident from the negative control WT-NPC protein extract.
4
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5
Figure 5: Attempts at ablating a polyclonal (puromycin-purified) line of CMV-NTR-NPCs using a
human codon preference usage-optimized form of the NfsB/NTR gene (144 silent mutations) referred to
as “HCO-NTR-NPCs”. Unlike the monoclonal line of CMV-NTR-NPCs (see Figure 3e-h), the HCO-NTR
construct did not contain an NTR-mcherry fusion protein, but only a GFP reporter gene as part of the piggyBac
transposon cargo (confirming successful NPC transformation). 1.0x106 HCO-NTR-NPCs were plated on a
10cm culture dish, and treated with 6mM of pre-dissolved, pharmaceutical grade Mtz through a media change.
The plate was imaged once daily at the 24-hour (a) and 48-hour (b) timepoints, and remaining cell density was
monitored qualitatively. Despite potential atrophy and “blebbing” of some cells at the 24-hour timepoint (a)
(circled in red), no major changes in cell density appeared to occur after 48 hours (b) of Mtz treatment. Scale
bars = 122µm.
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4) HSV-TK Plasmids and the Creation of HSV-TK NPCs
Due to the negative findings in selectively killing my line of hiPSC-derived NPCs with the
NTR-Mtz ablation system, an alternative approach using the HSV-TK + GCV/BVDU ablation
system was instead pursued- the first step of which entailed finding an appropriate gene transfer
vector (Research Aim 1). A two plasmid, Tol2 transposon-based gene transfer vector for HSV-
TK was selected from plasmids available from the Addgene repository. The Tol2 plasmids that
were selected consisted of: pCMV-Tol2 (Figure 6a) - containing the Tol2 transposase, and
pkTol2P-PTK (Figure 6b) - containing the actual Tol2 transposon carrying a puromycin-
resistance-HSV-TK fusion protein gene driven by a PGK promoter.
6a
Figure 6a: pCMV-Tol2 plasmid encoding the Tol2 transposase enzyme. Important
features of interest include an ampicillin resistance gene (top left in light green) for
bacterial selection, and a CMV-enhancer-promoter combination (top centre in white)
driving a Tol2 transposase gene (right side, downstream (below) the Kozak consensus
sequence (supporting eukaryotic translation)), and SV40 small T intron (mammalian gene
expression/transcription). pCMV-Tol2 was required for providing the Tol2 transposase
enzyme in order to excise the Tol2 transposon cargo (puromycin-resistance-HSV-TK
fusion protein gene- PTK) from plasmid pKTol2-PTK using the flanking Tol2
recognition sites and support integration into the genomes of target NPCs.
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6b
Figure 6b: pKTol2P-PTK plasmid encoding the Tol2 transposon and puromycin-
resistance-HSV-TK fusion protein gene (PTK). Important features of interest include a
kanamycin resistance gene (top left in light green) for bacterial selection, a Kozak consensus
sequence (supporting eukaryotic translation), and the Tol2 transposon itself carrying the PTK
gene (~500bp downstream/below primer Tol2-PTK-F1 (539-558) marked in purple) driven
by a PGK promoter. The main sequencing primers (marked in purple) indicating the relative
locations of each respective feature (~500bp downstream of each primer) were: Puro-F
(1244-1264, for the puromycin-resistance gene portion of PTK), mPGK-F (658-677, for the
PGK promoter), and Tol2-PTK-F1 (539-558, for the main portion of the Tol2 transposon
recognition sites and PTK gene).
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5) GCV Bystander Effect: Mixed HSV-TK & HCO-NTR-NPCs
After plasmid DNA purification, and confirmation through restriction digest and sequencing
(described in depth in Chapter 3: Materials and Methods- Section 4), the two plasmid, Tol2
transposon system (Figures 6a-b) was used to transform cells from the same original line of
hiPSC-derived NPCs (used to generate the CMV-NTR-NPCs described earlier) and purified
through puromycin. Bystander effects were evaluated as HSV-TK NPCs were plated along with
HCO-NTR-NPCs in 10cm culture plates, both separately (Figure 7a-b) and together at equal
ratios (Figure 7c) and treated with 1µg/mL GCV dissolved in media for 96 hours (three 10cm
plates total: HCO-NTR-NPCs + GCV (Figure 7a, e), HSV-TK NPC + GCV (Figure 7b, f), and
1:1 mixed HSV-TK and HCO-NTR-NPCs + GCV (Figure 7c,g ). A fourth control plate of
equal-ratio-mixed HSV-TK and HCO-NTR-NPCs (Figure 7d, h) was additionally maintained.
7
Figure 7: The evaluation of bystander effects of the HSV-TK + GCV ablation system in human-iPSC-
derived NPCs. 1.0x106 cells were plated on each of four separate, 10cm culture plates and subjected to
1µg/mL GCV (or regular media for the control plate) through media changes after 24 hours. Top (a-d) and
bottom row (e-h) images represent the 24-hour and 96-hour timepoints respectively. The cell composition of
each plate was as follows: (a&e) HCO-CMV-NTR GFP+ NPCs; (b&f) HSV-TK NPCs; (c&g) and (d&h)
(negative control) equal ratio-mixed HSV-TK and HCO-NTR-NPCs. Scale bars = 122µm (a-d), 125µm (e-h).
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After 96 hours of treatment with 1µg/mL of GCV, HCO-NTR-NPCs were as expected (since
they should be unable to phosphorylate GCV into its active, triphosphate form), relatively
unaffected (Figure 7e), while considerable cell death was observed qualitatively for both HSV-
TK NPCs (Figure 7f), and the mixed cells (Figure 7g). Especially noteworthy was the large-
scale death of GFP+, HCO-NTR-NPCs in the mixed cell population (Figure 7g), indicating the
obvious presence of a bystander cell killing effect for the HSV-TK NPCs after GCV treatment.
6) GCV/BVDU Bystander Effects: Mixed HSV-TK & WT-NPCs
8
Figure 8: The evaluation of bystander effects of the HSV-TK + GCV ablation system in hiPSC-
derived NPCs. 5.0x104 cells (1:1 mixed HSV-TK and GFP+ WT-NPCs) were plated on each well of two
separate, 6 well culture plates and subjected to increasing concentrations of GCV (0.0625-1µg/mL) through
media changes after 24 hours. The left half (a-f) represent the 0-hour timepoint, wherein GFP+ cells were
present in all wells, whereas the right half (g-l) represent the 96-hour timepoint, wherein considerable cell
death of both cell types (including virtually all GFP+ WT-NPCs) was observed for all GCV concentrations.
Scale bars = 99µm (a-c), 100µm (d-l).
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Due to the substantial bystander effects observed with GCV ablation of HSV-TK NPCs,
targeted cell elimination with an additional prodrug, BVDU, understood to exert a reduced
bystander effect upon ablation of HSV-TK-expresing cells, was pursued (reviewed in Dachs et
al., 2009). For the comparison of bystander effects between GCV and BVDU, two six well
culture plates were established wherein 5x104 cells consisting of equal ratio mixed HSV-TK
NPCS, and GFP+ WT-NPCs (also hiPSC-derived) were plated per well and subjected to
increasing concentrations (0.0625-1µg/mL) of either GCV (Figure 8) or BVDU (Figure 9).
9
Figure 9: The evaluation of bystander effects of the HSV-TK + BVDU ablation system in hiPSC-derived
NPCs. 5.0x104 cells (1:1 mixed HSV-TK and GFP+ WT-NPCs) were plated on each well of two separate, 6
well culture plates and subjected to increasing concentrations of BVDU (0.0625-1µg/mL) through media
changes after 24 hours. The left half (a-f) represent the 0-hour timepoint, wherein GFP+ cells were present in
all wells, whereas the right half (g-l) represent the 96-hour timepoint, wherein considerable cell death of both
cell types (including virtually all GFP+ WT-NPCs) was observed for all BVDU concentrations. Scale bars =
100µm.
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Both GCV and BVDU treatment of the mixed NPC populations for 96 hours (with all
concentrations) led to the noticeable depletion (in terms of remaining attached cells) of nearly all
GFP+ WT-NPCs (Figures 8-9). As such, a bystander effect was clearly apparent for both
prodrugs in combination with HSV-TK-expressing hiPSC-derived NPCs. I next attempted to
quantitatively evaluate the ablational/cell-killing efficiency of both the GCV and BVDU
prodrugs solely on HSV-TK NPCs (Figures 10-12).
7) GCV and BVDU Successfully Ablate HSV-TK hiPSC-NPCs
Figure 10: The ablation of proliferating hiPSC-derived HSV-TK+ NPCs with GCV (0.0625-
1µg/mL) for 96 hours. Four 6-well plates of 5.0x104 initial NPCs were set up and each well received
increasing concentrations of GCV (b-g). Quantification of attached cells/well was accomplished via
DAPI staining and peak analysis image processing in ImageJ, displayed above as: a) after 96 hours, h)
from 24-96 hours; statistical comparisons were performed via analysis of variance (ANOVA). Scale
bars = 100µm. Data was collected from the means of three replicate experiments (N=3).
10
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11
Figure 11: The ablation of proliferating hiPSC-derived HSV-TK+ NPCs with BVDU (0.0625-
1µg/mL) for 96 hours. Four 6-well plates of 5.0x104 initial NPCs were set up and each well received
increasing concentrations of BVDU (b-g). Quantification of attached cells/well was accomplished via
DAPI staining and peak analysis image processing in ImageJ, displayed above as: a) after 96 hours,
h) from 24-96 hours; statistical comparisons were performed via analysis of variance (ANOVA).
Scale bars = 100µm. Data was collected from the means of three replicate experiments (N=3).
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Figure 12: Comparison of the ablation of hiPSC-derived HSV-TK+ NPCs with GCV and BVDU
(0.0625-1µg/mL for both) after a) 96 hours, and b) 24-96 hours. Eight (4 per prodrug) 6-well
plates of 5.0x104 initial NPCs were set up and each well received increasing concentrations of
GCV/BVDU. Quantification of attached cells/well was accomplished via DAPI staining and peak
analysis image processing in ImageJ; statistical comparisons were performed via ANOVA. No
significant differences were detected between a) equal concentrations of GCV & BVDU after 96
hours, nor b) equal concentrations of GCV & BVDU across time points.
12
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Quantification of the ablational efficiencies of both GCV and BVDU was set up as described in
Chapter 3: Materials and Methods and displayed above in Figures 10-12. After 96 hours of
GCV treatment (Figure 10a-g), all concentrations used were found to produce significant
(p<0.01) differences (detected via ANOVA) over the control well in terms of the mean estimated
number of remaining attached cells. BVDU ablation produced similar results (Figure 11a-g),
albeit slightly more gradual, as the lowest concentration used (0.0625µg/mL) did not produce a
significant difference in remaining attached cells over the control. The 1 µg/mL concentration of
both prodrugs resulted in a reduction in attached cells of over 80% compared to controls
(Figures 10a &11a). No significant differences were detected in the estimated number of
attached cells between 96-hour treatments of GCV and BVDU (Figure 12a). Similarly, no
significance was detected comparing the quantities of attached cells of each prodrug’s treatments
individiually across timepoints (Figures 10h and 11h), nor for comparing the same treatment
concentration of both prodrugs across timepoints (Figure 12b).
As the cells were proliferative, even in the highest concentration of each prodrug used (1µg/mL),
there were still many more cells remaining than initially plated (~2x105 vs 5 x104) however the
proliferative ability was certainly significantly reduced compared to controls (1.2-1.4 x106cells
remaining). Additionally, as the method used for cell quantification only took into account
attachment to each well, and did not assess viability, it is considerably possible that the vast
majority of cells remaining in the wells of the highest concentrations of each prodrug treatment
were in fact non-viable (possibly undergoing apoptosis), as may be discerned from Figures 10g
and 11g.
The final step in assessing GCV and BVDU ablation lay in the elimination of a potential
confounding variable- namely that of DMSO, the solvent in which both prodrugs had been diluted
in prior to mixing with the culture media used for the experiments in Figures 10-12. In order to
determine that the cell-killing/proliferative inhibition effects seen for both GCV and BVDU had
not been caused by DMSO cytotoxicity, a minor experiment was set up whereby 5.0x104 HSV-TK
NPCs were plated in each of two wells of a 6-well plate, and after 24-hours, subjected to media
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changes containing either 0.4% dissolved DMSO (the highest concentration used for GCV/BVDU
dilution) (Figure 13c), or 0.4% dissolved PBS (Figure 13a). Each well was imaged at the starting
(0-hour) and 48-hour timepoints to obtain a general qualitative determination of any potential
DMSO-related toxicity. No obvious discernable effects on cell density/proliferation were detected
with 0.4% DMSO treatment of HSV-TK NPCs (Figure 13d).
13
Figure 13: Assessing the potential for DMSO cytotoxicity with HSV-TK NPCs.
5.0x104 HSV-TK-NPCs each were plated in two wells of a 6-well culture plate and
after 24-hours subjected to media treatments containing: a) 0.4% PBS, or c) 0.4%
DMSO. After 48 hours of treatment (b and d), the wells were imaged, and general cell
density was assessed qualitatively. Both the treatments of 0.4% PBS (b) and 0.4%
DMSO (d) appeared to have no effect on the general proliferation of HSV-TK-NPCs.
Scale bars = 100µm.
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Chapter 5: General Discussion, Future Directions & Conclusion
Overall, a simple, “proof of concept” has now been demonstrated for the successful ablation of
hiPSC-derived NPCS with the HSV-TK + GCV/BVDU ablation system through the preceeding
work in Chapter 4: Results. Numerous challenges and pitfalls were overcome throughout the
course of my MSc project and I shall attempt to address and elaborate on the most germane and
critical points relating to each experiment, as well as the broader extrapolations of my work in
relation to the grander picture within which it should be framed. There are numerous
possibilities for building upon my work and various avenues and directions to embark on for
future investigations.
5.1 Summary of HSV-TK+GCV/BVDU Results
The most critical results obtained in this project are the effective ablation/proliferation reduction
of proliferating human iPSC-derived NPCs using the HSV-TK +GCV/BVDU ablation system.
A reduction in the estimated total number of attached cells of 80% or greater over controls was
detected for both the GCV and BVDU prodrugs at the highest concentrations used (1µg/mL)
after 96 hours (Figures 10a &11a). As mentioned previously in Chapter 5: Results, no
significant differences were detected between equivalent concentrations of the GCV and BVDU
prodrugs after 96 hours of treatment in terms of the estimated quantity of attached HSV-TK
NPCs per well of each plate (Figures 10h and 11h). Additionally, no significant differences in
regards to the estimated number of remaining attached cells were detected among the four-day
time points for equivalent concentrations of either GCV and BVDU alone, nor for GCV and
BVDU compared together (Figures 10h, 11h, 12b).
In terms of bystander effects, the obvious presence of one was first detected for GCV when
HSV-TK NPCs were cultured together with HCO-NTR NPCs (Figure 7g), as the vast majority
(e.g., all but 2 cells in the representative image depicted) of GFP+ HCO-NTR NPCs no longer
remained attached to the plate after four days of 1µg/mL GCV treatment. Additional bystander
effects were detected after 96 hours for all concentrations of GCV employed (0.0625-1µg/mL)
from the combined equal culture of HSV-TK NPCs and GFP+ WT-NPCs (Figure 8h-l). All
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BVDU treatments (0.0625-1µg/mL) similarly to GCV, also displayed bystander effects after 96
hours of treatment (Figure 9h-l), as identical to GCV, they also resulted in the elimination of all
GFP+ WT-NPCs.
5.2 HSV-TK+GCV/BVDU Ablation in Context
Concerning the HSV-TK+ GCV/BVDU ablation system, my results have indicated the existence
of a bystander effect for both prodrugs when utilized to ablate hiPSC-derived, HSV-TK-
transformed NPCs (as indicated by Figures 7g, 8h-l, and 9h-l). My personal findings, although
only rudimentary and qualitative with relation to bystander effect, nevertheless are partially as
expected by literature, as both prodrugs are known to possess some degree of bystander effect
(due to GCV-monophosphate and BVDU-diphosphate “spillover” to neighbouring cells through
Cx43 channels); however, it was not possible to detect an obvious difference between the
bystander effects of GCV and BVDU (reviewed in Dachs et al., 2009).
Although BVDU was predicted to possess a reduced bystander effect over GCV, such an
investigation was deemed to be beyond the scope of this project, and thus future studies will be
necessary for firmly establishing any significant differences in prodrug bystander effects in
regards to ablating hiPSC-derived HSV-TK NPCs (reviewed in Degrève et al., 1999).
As mentioned briefly in Chapter 4: Results, Section 7, many if not most of the remaining
attached cells may not have been viable, and either in the process of, or recently underwent
apoptosis, and simply not yet detached (possibly inferred from Figure 10e-g and 11e-g). No
significant differences were detected in terms of estimated attached cell number between
equivalent treatments of GCV and BVDU (Figure 12), and thus although a difference may exist
between the ablational efficiencies of each prodrug, my personal experiments could not discern
any. Perhaps future studies employing wider prodrug concentration ranges and longer
timepoints may be able to determine if any ablational efficiencies exist between GCV and
BVDU specifically for hiPSC-NPCs.
Although the mechanisms of apoptosis induced by GCV and BVDU are indeed understood to be
similar (as both are nucleotide analogs inducing apoptosis primarily through incorporation into
replicating genomic or mitochondrial DNA causing chain-termination), there are most certainly
minor, yet perhaps critical differences between these mechanisms, such as BVDU’s ability to
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target thymidylate synthase, and GCV’s ability to induce extrinsic, Fas-pathway apoptosis
(involving DISC-formation) without the activation of Fas ligand (Balzarini et al., 1987; Beltinger
et al., 1999). These aforementioned differences in the apoptotic pathways induced for GCV and
BVDU could play a role in ablational efficiency differences, if any are yet detected for hiPSC-
derived NPCs.
Although significance was determined for the difference in the estimated number of attached
cells remaining after 96 hours for all treatments of GCV (Figure 10a) and for all but the lowest
concentration of BVDU (Figure 11a), there were still large quantities of cells remaining
(~2x105), even for the highest concentrations used. Since the HSV-TK NPCs were cultured and
treated with GCV and BVDU in SF9 media, they were certainly proliferative, and it is possible
that the concentrations of GCV/BVDU used may not have been completely sufficient to
overcome proliferation and accomplish both cell death and detachment from wells for all HSV-
TK NPCs. Such a result is understandable considering the previous reported difficulties in
achieving complete ablation and removal of proliferating NPCs (alebit ones of ESC origin) with
the HSV-TK+GCV ablation system (Tieng et al., 2016).
5.3 Advantages and Challenges of HSV-TK+GCV/BVDU Ablation
The proof of concept tool for the ablation of hiPSC-derived NPCs using the HSV-
TK+GCV/BVDU system herein demonstrated offers several critical advantages for further
usages, along with the caveat of significant challenges. Through the application of 1µg/mL of
either GCV or BVDU for a time duration of 96 hours, it is possible to reduce the number of
remaining cells in vitro by at least 80% over controls, allowing for targeted cell death of
proliferating NPCs at the very least. For future in vivo usages, effective concentrations and
timepoints established will likely require modification, however as a GCV dosage of
100mg/kg/day has previously been shown to effectively ablate GFAP+ endogenous murine
astrocytes without resulting in any serious general toxicity related side effects, reaching a similar
internal GCV concentration as demonstrated in my in vitro work should likely be achievable
without serious toxicity related side effects (Bush et al., 1998).
Both GCV and BVDU are understood to be able to cross the blood brain barrier/blood spinal
cord barrier and are additionally FDA approved substances, allowing for potential future clinical
usages of either prodrug, if it is deemed effective and necessary to employ them for controlling
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the proliferation of transplanted hiPSC-derived NPCs in order accomplish improvements in
terms of physical repair and functional recovery (Tieng et al., 2016; reviewed in De Clercq
2005). Additionally, while GCV is considered a potential carcinogen and posses a number of
potentially serious side effects, such as anaemia, nausea, vomiting, diarrhea, headaches, and
confusion, BVDU offers the advantage of carcinogenic-free, and relatively side-effect free
ablation, with only mild nausea as an uncommon adverse reaction (reviewed in Zhang et al.,
2015; reviewed in De Clerq 2005).
Two major, yet certainly not insurmountable challenges exist for the HSV-TK+GCV/BVDU
ablation system with the targeted elimination of hiPSC-derived NPCs. These challenges are
specifically: (1) the presence of an obvious bystander cell-killing effect, and (2) the potential
difficulty in ablating non-proliferating/post-mitotic cells. The bystander effect for both the GCV
and BVDU prodrugs as observed in the results of this thesis (Figure 8h-l & 9h-l) presents an
obstacle in terms of the selectivity of ablation- (e.g., when employed for in vivo SCI
neuroregeneration studies with transplanted HSV-TK hiPSC-derived NPCs, will potential side-
effects occur and will neurorepair be hampered due to the unwanted death of neighbouring
cells?). The bystander effects for both the GCV and BVDU prodrugs are understood to be
dependent on Cx43 gap junction expression, cell type, and proximity among other factors
(reviewed in Dachs et al., 2009; reviewed in Degrève et al., 1999).
Realistically, a bystander effect may be unavoidable in HSV-TK+GCV/BVDU ablation,
however as previous studies employing both prodrugs have demonstrated successful targeted
ablation in vivo of neural cell types without obvious adverse reactions due to bystander effects,
achieving similar results should theoretically not be an overly arduous task (Tieng et al., 2016;
Field et al., 1984). As BVDU ablation of HSV-TK hiPSC-derived NPCs has been demonstrated
in this thesis to be as effective GCV ablation, and as BVDU has been previously demonstrated to
exert a greatly reduced bystander effect, the challenge of bystander effects in vivo could partially
be mitigated, albeit not removed entirely through the use of BVDU instead GCV (reviewed in
Dachs et al., 2009; reviewed in Degrève et al., 1999). Additionally, bystander effects for in vivo
transplantations of my HSV-TK hiPSC-derived NPCs could be potentially be further reduced
through the administration of substances known to block Cx43 channels, such as 18 alpha-
glycyrrhetinic acid or 1-octanol (Princen et al., 1999).
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Concerning challenge (2): the potential difficulty in ablating non-dividing/post-mitotic cells
(e.g., NPC-derived neurons), increasing the dosage of GCV/BVDU should theoretically allow
for effective cell ablation through the incorporation of GCV/BVDU-triphosphate into replicating
mitochondrial DNA during mitochondrial turnover, and the resultant induction of the intrinsic,
mitochondrial apoptotic pathway (Beltinger et al., 1999, Laberge et al., 2013). As previous
research has achieved successful ablation of HSV-TK expressing, senescent human fibroblasts
using mildly increased concentrations of GCV, and confirmed that it occurred through the
mitochondrial pathway via the detection of mitochondrial death signals, it would in the opinion
of this author, be a fair deduction that such should be accomplishable for targeting post-mitotic
neurons differentiated from transplanted hiPSC-derived HSV-TK NPCs (Laberge et al., 2013).
Nevertheless, if the concentrations required for achieving post-mitotic cell ablation in vivo in SCI
model rats is determined to be in excess of the general toxicity range (e.g., greatly in excess of
100mg/kg/day), then the ablation tool will still be effective for controlling transplanted hiPSC-
derived NPC proliferation (overarching question (3) from Chapter 2: Research Aims), thus
allowing for studies to occur in which the optimal amount of proliferation/NPC quantity required
for effective neuroregeneration may be determined.
5.4 Reflections on NTR-Mtz Ablation Approaches
With regards to the sub-cloning of each of the NTR-inserts into the piggyBac transposon plasmid
vector, considerable difficulties were encountered initially, largely believed to be inherent to the
large sizes of all three inserts, as well as the target vector plasmid. As the early attempts at NTR-
piggyBac subcloning involved the use of blunt-ends (no compatible 5’ or 3’ overhangs), relying
merely on random collisions of molecules for ligation of insert and vector, it is understandable
for the continued lack of success in obtaining positive clones, considering that even the smallest
NTR insert fragment (CMV-NTR) was over 2kb in size. Previous research has indicated similar
difficulties with regards to blunt-end subcloning of transgenes into gene transfer vectors wherein
both inserts and vectors were of such aforementioned (2-3kb for inserts, 10kb+ for vectors)
(reviewed in Kantor et al, 2014; reviewed in Manfredsson et al., 2016).
Even with the initial “eureka-moment” successes of obtaining positive NTR-piggyBac clones
through PCR-amplification adding MluI-restriction sites for compatible end cloning of the NTR
inserts and piggyBac vector, additional steps and verifications were required, in the form of
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further restriction digests to remove the unrelated insert, which itself proved to be a more
difficult task than previously expected, as self-ligation of the linearized, NTR-piggyBac
plasmids, or less likely, successful transformation of DH10β E. coli also required multiple
attempts for success. Although in hindsight, possibly additional planning and experimental
design of the overall strategic steps for NTR-piggyBac subcloning may have considerably
shortened the timeframe with which that section of my MSc project elapsed, the strategies and
steps that were pursued nevertheless provided valuable skills and experience.
Additionally, despite the negative findings with the Mtz ablation of CMV-NTR-NPCs created
through the use of the subcloned CMV-NTR-piggyBac plasmid, the considerable effort and time
spent creating the plasmid through extensive molecular biology should not perhaps be considered
wasted, as in addition to providing the opportunity for learning techniques, it created a safe, and
efficient gene-transfer vector containing a useful suicide gene for potential future attempts at
achieving success with the NTR-Mtz ablation system of hiPSC-derived NPCs or other human
neural cell types in the broader context of neuroregeneration loss-of-function studies (reviewed
in Kantor et al., 2014). As there have yet to be any human or other mammalian neural cell
reported usages of the NTR-Mtz ablation system, the plausible future success of the system with
the targeted killing of hiPSC-derived NPCs would certainly still be a novel and noteworthy
technique (reviewed in Williams et al., 2015).
Gene expression of NTR/NfsB in either the CMV-NTR-NPC monoclonal line, or HCO-NTR-
NPC polyclonal line was not assessed during the course of my MSc work, and thus may be a
potential culprit for the lack of Mtz ablational success demonstrated. In hindsight, rather than
pursuing the possible issue of codon-optimization, the hurdle for which HCO-NTR-NPCs were
created as an attempt to solve, a more immediate solution of investigating NTR/NfsB gene
expression should likely have been taken, which would have theoretically been simple through
quantitative real-time PCR (qRT-PCR). The obstacle towards NTR-Mtz ablational success may
have also been located downstream, in the form of protein expression, however there is currently
no readily available, commercial antibody for NfsB, and thus Coomassie blue total protein
staining of CMV-NTR-NPC protein extract was pursued instead. Despite the possible presence
of a faint band (Chapter 4: Results, Section 3, Figure 4), it is not immediately obvious that
NTR protein expression was present, as not only could the band represent a different protein of
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equivalent size, the control sample still showed a blurry/very faint band at the same mass of
~50kDa, obfuscating any certainty of detected NTR protein.
As mentioned briefly earlier in Chapter 3: Materials and Methods, Section 2, targeted
transformation (such as using Gateway cloning® (ThermofisherScientific)) of hiPSC-derved
NPCs with the promoter-NTR constructs may have been a more suitable strategy for pursuing
success, as previous work has indicated that transgene stability is considerably increased
(maintaining genomic integration and transcription over multiple passages) through selective
integration of transgenes into cell-life-vital genes and regions (reviewed in Kantor et al., 2014;
reviewed in Manfredsson et al., 2016; reviewed in Joshi et al., 2017).
If the lack of NTR/NfsB gene/transcript expression was the primary reason behind the apparent
lack of Mtz-mediated ablational success for CMV-NTR NPCs, such a process may have
occurred through repression, silencing or interference (e.g., through methylation, etc.) of the
integrated NTR transgene from NPC genomic elements, which may still be possible despite
multiple piggyBac transposon insertions (~15) likely occurring on average per electroporated
NPC, due to the lack of targeted insertion within cell-life-dependent genes (reviewed in Zhao et
al., 2016; reviewed in Li et al., 2013; reviewed in Grabundzija 2010; reviewed in Wu et al.,
2006). Interference with NTR transgene expression in CMV-NTR NPCs may even have
occurred as a result of the piggyBac transposon’s inherent preference for integration within
transcriptional regions, or due to promoter repression from the minor transcriptional activity of
the piggyBac 5’LTR (reviewed in Vargas et al., 2018; Gogol-Döring et al., 2016; Cadiñanos and
Bradley, 2007).
The strategy of pursuing human codon optimization for NTR expression perhaps should not have
been the immediate first or second choice for troubleshooting the NTR-Mtz ablation system, as
gene transcript expression of CMV-NTR NPCs, and transforming additional cell types as
controls may likely in hindsight have been more efficient and relevant choices for effective NTR
troubleshooting. If an additional cell type had been transformed with the CMV-NTR-piggyBac
plasmid and ablation pursued with Mtz, the possibility of an NPC-specific issue (e.g., resistance
due to exceptionally rapid proliferation, etc.) preventing ablation could perhaps have been
discovered or ruled out. Perhaps issues relating to high NPC proliferation of my hiPSC-NPC
96
line (especially in serum free hormone mix/SF9 media) or prodrug uptake may have been the
answer to the enigma of unsuccessful ablation.
The concentrations of Mtz (up to 10mM) and timepoints (up to 96 hours) were more than has
been demonstrated sufficient with various zebrafish and frog cell types, yet it is possible that
human-specific differences may have warranted higher concentrations or longer timepoints
(reviewed in Williams et al., 2015; reviewed Sekizar et al., 2015; reviewed in Chung et al.,
2013). Solubility of the Mtz prodrug was considered to be a potential obstacle to ablation,
however it should likely be ruled out, as the ablation of HCO-NTR-NPCs was attempted using a
pre-dissolved, pharmaceutical intravenous sack of Mtz solution. Although potential apoptotic
“blebbing” or atrophy appeared to have been detected after 24 hours of Mtz treatment in HCO-
NTR-NPCs (Figure 5a), after 48 hours no obvious indications of reduced cell density/cell death
were apparent (Figure 5b), indicating that for whichever reason of any of the above stated
(transgene repression, translational issues, cell-type-dependent Mtz resistance due to rapid
proliferation) HCO-NTR-NPC ablation with Mtz had proven more challenging than originally
forseen.
5.5 Potential Applications and Future Directions of HSV-Tk+GCV/BVDU hiPSC-Derived NPC Ablation
There are numerous possibilities for potential applications and future directions of the HSV-TK
hiPSC-derived NPCs developed in this thesis. An obvious usage as outlined earlier is the in vivo
ablation of the cell line post-transplantation in SCI rats at different time points (e.g.,
administering GCV/BVDU at 2h, 12h, 24h, 48h, 72h, 96h post transplantation and 14 days post-
injury etc.) and comparing the changes in physical repair (as could be detected via histology
using human nuclear antigen as a potential marker of transplanted cells) and functional recovery
(as detected via behavioural tests- e.g., catwalk, grip strength, etc.) to animals receiving the same
hiPSC-derived NPC transplantions without ablations. Bystander effects could theoretically be
detected in vivo through Western blots comparing apoptosis markers (e.g., caspase 3 etc.) from
spinal cord sections of both ablated and non-ablated, hiPSC-NPC transplant animals.
Although the HSV-TK+GCV/BVDU ablation approach developed in this thesis used only a
constitutive PGK promoter allowing for ablation of all transplanted cell types, through
subcloning work, cell-type specific promoters (e.g., DCX, MBP, GFAP, etc.) could be added
97
instead and thus potentially allow for cell-type-specific ablation (e.g., neurons vs.
oligodendrocytes vs. astrocytes). Such a cell-specific promoter-based approach could potentially
allow for pursuing overarching question (1) (from Chapter 2: Research Aims) in regards to the
effectiveness of oligodendrocytes/remyelination vs. neurons/synaptic re-establishment in
achieving SCI physical repair and functional recovery. Furthermore, the HSV-TK hiPSC-
derived NPCs developed in this thesis could theoretically be additionally employed for pursuing
neuroregeneration studies for other (non-SCI) CNS disorders/conditions/diseases that could
benefit from cell transplantation therapies (e.g., stroke, Parkinson’s, multiple sclerosis, epilepsy
etc.).
An immediately obvious direction for further evaluation of hiPSC-HSV-TK NPC ablation with
GCV and BVDU would be assessing not only the remaining cell density after treatment, but also
the viability of remaining cells, such as could be pursued through MTT assays (discussed in
Chapter 1, Section 3.1.2), or live-dead assays with a combination of markers including calcein
AM (for live cells, due to the requirement for cellular esterase activity to produce a fluorescent
metabolite) and propidium iodide (for dead/non-viable cells due to lack of membrane
permeability in most live cells).
Quantitative differences in bystander effects of GCV and BVDU in ablation of hiPSC-derived
NPCs could likely be accomplished through continuing with the strategy that I had initially
pursued- FACS of both live and dead NPCs after GCV/BVDU treatments at specific timepoints.
The quantitative results of this thesis (Figures 10-12) could indeed be useful for further studies
of GCV and BVDU bystander effects, as effective concentrations and timepoints for ablation
(likely a minimum of 1µg/mL for a minimum of 4 days, the highest timepoint and concentration
used) have already been determined for hiPSC-derived HSV-TK NPCs.
Furthermore, for fine-tuning of hiPSC-HSV-TK NPC ablation, it would likely be necessary to
fully confirm both gene/mRNA transcript expression and protein translation of HSV-TK, which
could be achieved through qRT-PCR and Western blotting respectively. Unlike the NTR/NfsB
protein, HSV-TK does indeed have commercially available antibodies, enabling the possibility
of evaluating and confirming HSV-TK protein expression for the hiPSC-HSV-TK NPC line
created over the course of this MSc project. Determining if the cell death observed in
GCV/BVDU ablation of hiPSC-HSV-TK NPCs is indeed occurring from apoptosis as expected,
98
and not necrosis would presumably be yet another important potential future direction, and as
such could be investigated through Western blot antibody staining of apoptotic markers, such as:
caspase 3, Bcl-2, cytochrome C, or annexin V (Ma et al., 2017; Zhuo et al., 2016).
As the HSV-TK Tol2 transposon gene-transfer vector system used did not possess a fluorescent
reporter gene, it could be potentially useful for future uses of hiPSC-HSV-TK NPCs to subclone
in a GFP gene for confirmation of successfully transformed NPCs, or even an mCherry gene for
the creation of a puromycin-resistance-HSV-TK-mCherry tri-fusion protein-encoding gene to
allow for visual identification of HSV-TK-expressing NPCs; however mChery fluorescence
should not perhaps be used as the sole indicator of HSV-TK expression, due to the challenges
faced with ablational success of CMV-NTR-NPCs, which similarly were expected to express
(yet may not have) NTR due to visual mCherry expression (faintly visible in Figure 3).
In terms of novelty, although the HSV-TK + GCV enzyme-prodrug ablation system has been
utilized extensively for decades, and has previously been used to target such neural cell types as:
C17.2 immortalized mouse cerebellar NPCs; C6 rat gliomas; GFAP-expressing mouse dNSCs;
and, even human ESC-derived NPCs, there have not currently been any reported usages of the
system for ablating hiPSC-derived NPCs (Tieng et al., 2016; Pu et al., 2011; Uhl et al., 2005; Li
et al., 2005; Morshead et al., 2003). Additionally, there have only been two reported usages of
the HSV-TK+GCV ablation system in an SCI context, specifically from studies that ablated:
endogenous murine NG2+ pericytes and OPCs, and murine GFAP+ reactive astrocytes (Hesp et
al., 2018; Faulkner et al., 2004). BVDU-mediated ablation of hiPSC-derived NPCs is further
considerably even more novel than the use of GCV for the same cell type, as apart from the
treatment of herpetic encephalitis for neurons of both mouse and human origin, there have not
currently been any reported uses of HSV-TK+BVDU ablation for other human neural cell types,
including NPCs (Field et al., 1984; Wigdahl et al., 1984; Wigdhal et al., 1983).
Ultimately, hiPSC-derived HSV-TK NPCs of the line developed in this MSc project should be
used for in vivo ablations after NPC transplants post-SCI in rodent models, which as described in
Chapter 2: Research Aims, is the larger picture within which my own MSc project is framed.
Perhaps through ablation of transplanted HSV-TK NPCs in SCI-model rats, questions (2) and (3)
of the primary unanswered questions with relation to NPC transplantation for SCI
neurorepair/neuregeneration, specifically the issues of: (2) short-term trophic
99
support/immunomodulation vs. long term migration, differentiation, and integration; and (3) the
amount/timing of proliferation required to occur for transplanted hiPSC-derived NPCs in order to
yield sufficient NPC and NPC-derived cell quantities (or quantities of trophic/immune-
modulating factors) to provide effective SCI physical repair and functional recovery, may yet be
at least partially answered.
It may be pertinent to perform a transcriptomic analysis on the HSV-TK NPC line developed in
this thesis in terms of the gene expression for trophic factors (e.g., BDNF, GDNF, CNTF, etc.)
at various timepoints when subjected to general differentiation conditions as would be
experienced in vivo. Such an experiment may allow for a clearer understanding of the levels of
various trophic factors expressed at various timepoints (e.g., 24h, 48h, 72h, 96h, 1 week, 2
weeks, and finally 3 weeks, etc.) of NPC proliferation and differentiation of the HSV-TK hiPSC-
derived NPC line, which could then assist in determining the most appropriate time to ablate
cells without losing significant trophic/immunomodulatory factor secretion. Additionally, it
would be vital to determine the conversion ratio between in vivo dosage (e.g., 100mg/kg/day
etc.) and the final internal concentration of GCV/BVDU that accumulates resultantly in the
injured cord niche.
As GCV is known to remain stable for up to 10 days, such a task may be achievable (Sachewsky
et al., 2014). Subsequently, in vivo ablations of the HSV-TK hiPSC-derived NPCs post
transplantation in SCI rodents could be performed with administrations of both GCV and BVDU,
using a final concentration range of 0.125µg/mL (the second lowest concentration used of each,
and the lowest concentration showing ablational significance over controls for both prodrugs) up
to 10µg/mL (as such higher concentrations have previously been used to ablate human ESC-
derived NPCs without detectable general toxicity) at timepoints ranging from 24h-post
transplantation up to 3 (or even 6) weeks post-transplantation. As iterated previously,
sensorimotor behavioural changes (reductions in functional recovery) could then be detected via
behavioural test comparisons to animals receiving NPC transplantations without ablations.
Similarly, alterations in the physical repair (e.g., remyelination, endogenous axonal regrowth or
neuronal replacement from transplanted hiPSC-derived NPCs and subsequent synaptic plasticity
reestablishment, etc.) could be detected via performing spinal cord ultra-microtome sectioning
and histology with immunohistochemical staining for for oligodendrocytes (e.g., MBP), and
neurons (e.g., TuJ1, etc.). Differences in synaptic rewiring between transplantation+ablation vs
100
transplantation-alone animal cords could more specifically be detected via pseudorabies virus
tracing in tract regions (e.g., cortical, dorsal, rubrospinal tracts etc.) adjacent to the injury level
(e.g., C7-T1 injury).
Although the HSV-TK +GCV/BVDU ablation system is expected to be effective at ablating even
non-dividing/post-mitotic cell types, such as mature neurons, through disruption of
mitochondrial DNA replication, in the event that such a process is deemed impractical for
hiPSC-derived NPCs in rodent model SCI usage (perhaps due to the necessary prodrug
concentrations possibly approaching the upper limit of general toxicity), the ablation in vitro
proof of concept demonstration that I have developed could still be used to assess the
aforementioned overarching question (3) (e.g., required amount/timing of transplanted iPSC-
derived NPC proliferation).
Likewise, through future subcloning work to add the DCX and MBP promoters to plasmid
pKTol2P-PTK (carrying the Tol2 transposon and puromycin resistance-HSV-TK fusion protein
gene) in place of the present PGK promoter to allow for selective ablation of early neurons and
myelinating oligodendrocytes respectively (as planned for NTR-Mtz ablation), question (1) in
regards to NPC transplants for SCI neuroepair/regeneration namely the issue of remyelination
versus neuronal regeneration/synaptic connectivity reestablishment (addressed in Chapter 2:
Research Aims) may additionally be elucidated. The three research aims of Chapter 2, namely:
(1) acquiring an effective gene-transfer vector for a suicide gene (originally NTR, and later
HSV-TK), (2) generating a cell line of suicide-gene expressing hiPSC-NPCs; and (3) performing
drug-directed ablations (Mtz originally and later GCV/BVDU) were in the opinion of this author,
realistic and reasonable, especially considering the difficulties and challenges faced with
achieving cell death for the NTR-Mtz ablation system.
101
5.6 Conclusion
In summary, I have generated a rudimentary proof of concept study of the HSV-TK +
GCV/BVDU ablation system in relation to hiPSC-derived NPCs. A set of three primary research
aims, namely: (1) acquiring an effective gene-transfer vector for a suicide gene (originally NTR,
and later HSV-TK), (2) generating a cell line of suicide-gene expressing hiPSC-NPCs; and (3)
performing drug-directed ablations (Mtz originally and later GCV/BVDU) were developed and
subsequently accomplished. Both the GCV and BVDU prodrugs have been shown to
significantly reduce the number of attached cells remaining after 96-hour treatments with
concentrations up to 1µg/mL (<80% reduction over controls for the highest concentrations used).
I was unable to detect any significant differences between prodrugs in regards to ablational
efficiency, nor in terms of the magnitude of bystander effects, however the presence of one was
obviously discernable for both prodrugs.
Initially the NTR-Mtz ablation system, with constructs involving three promoters, for not only
general NPC ablation, but also targeted ablation of early neurons and myelinating
oligodendrocytes was considered and pursued, however due to challenges and difficulties in
achieving cell death with the NTR-Mtz system, the goals and aims of my MSc research project
were narrowed to the more reasonable and realistic approach of providing initial evaluation of
solely hiPSC-derived NPC ablation. Considerable future work beyond the scope of my own
MSc project will be necessary to evaluate GCV and BVDU-mediated ablation of hiPSC-derived
NPCs, including cell viability determinations and, further bystander effect evaluations, however
HSV-TK-expressing hiPSC-derived NPCs may yet prove successful for examining the larger
picture within which I have attempted to frame their future intended uses, concerning the effects
of NPC-transplant-mediated neurorepair and regeneration for future SCI therapeutic treatment.
102
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Appendix I: List of Contributions
Dr. Mohamad Khazei: Providing the hiPSC-derived NPC line, as well as assistance with
learning many cell culture techniques and general guidance with regards to strategies for my
overall MSc project.
Dr. Chris Ahuja: Providing advice and suggestions for developing the monoclonal NTR-NPC
line, as well as the human-codon-optimized (HCO)-NTR-NPC cell line.
Dr. Mahmood Chamankhah: Aiding in developing the strategies for successful NTR-piggyBac
subcloning, as well as guiding and mentoring with subcloning experiments
Jian Wang: Additional assistance with learning and improving cell culture techniques
Priscilla Chan: Assisting with providing a protocol for the steps involved in NTR protein
extraction, quantification, and protein staining.
James Hong: Performing “peak analysis” image processing for quantification of remaining
attached cells in the context of HSV-TK NPC ablation experiments (Figures 10-12).