CHEMICAL AND ELECTRICAL STIMULATION IN ENHANCING
HUMAN NEURAL STEM CELL MIGRATION
A Project
Presented to the faculty of the Department of Biological Sciences
California State University, Sacramento
Submitted in partial satisfaction of
the requirements of the degree of
MASTER OF ARTS
in
Biological Sciences
(Stem Cell)
by
Michelle So
SPRING
2012
ii
© 2012
Michelle So
ALL RIGHTS RESERVED
ii
CHEMICAL AND ELECTRICAL STIMULATION IN ENHANCING
HUMAN NEURAL STEM CELL MIGRATION
A Project
By
Michelle So
Approved by:
_____________________________________, Committee Chair
Thomas Landerholm, Ph.D.
_____________________________________, Second Reader
Jan Nolta, Ph.D.
_____________________________________, Third Reader
Christine Kirvan, Ph.D.
_____________________________________
Date
iii
Student: Michelle So
I certify that this student has met the requirements for format contained in the University format
manual, and that this project is suitable for shelving in the Library and credit is to be awarded for
the project.
_______________________________, Graduate Coordinator
Ronald M. Coleman, Ph.D.
Department of Biological Sciences
iv
___________________
Date
Abstract
of
CHEMICAL AND ELECTRICAL STIMULATION IN ENHANCING
HUMAN NEURAL STEM CELL MIGRATION
By
Michelle So
Studies of multipotent neural stem cell (NSC) transplantations demonstrate their potential as a
regenerative therapy to improve functional recovery following neurotrauma. However a major
obstacle hindering greater tissue regeneration is the inefficient rate of engraftment and migration
of transplant cells to the site of injury. Towards this challenge of improving NSC migration,
other studies have discovered chemotactic or motogenic properties of certain trophic cytokines
that are important for maintaining NSC multipotency. However fewer studies have investigated
the use of physical migration cues, specifically direct current electric fields (EF) and even fewer
look at the migratory responses of a combined chemical-electrical migration stimulation
condition. In this study we compared the migration responses of human embryonic stem cell
(ESC) line, H9, derived NSCs to chemical and electrical migration stimulation combinations in
vitro. Our results suggests that trophic cytokines such as basic fibroblast growth factor (bFGF),
epidermal growth factor (EGF) and adenosine triphosphosphate (ATP) traditionally known to
induce a chemotactic response, did not have a positive chemokinetic effect on our H9-NSC
migration. Conversely, exposure to direct current electric field stimulation enhances migration
by increasing motility, migration directionality and distance travelled in a linear, less tortuous
path. We also observed preliminary data that suggests contrary to intuitive expectations,
v
electrical and EGF combined stimulation did not additively increase cell motility significantly or
for any extended period of time. Interestingly, the co-stimulation appears to increase
directedness of electrotactic migration. This suggests that other motogenic cytokines and
electrical co-stimulation may positively enhance hNSC motility and directionality in vitro. It also
suggests the combination could have similar effect on NSC migration if applied in vivo and
warrants further study towards optimizing stimulation conditions. However because EF
stimulation is the major contributing factor behind the increase in motility and linearity of
migration path, the addition of a chemical component to optimize electrotaxis must be carefully
considered due to complicated regulation behind receptor activation and downstream
intracellular cascade signaling.
____________________________, Committee Chair
Thomas Landerholm, Ph.D.
____________________________
Date
vi
ACKNOWLEDGEMENTS
There are few privileged scholars who have the luxury of opportunity, mentorship and
security. I am honored to have been given the unique opportunity by Bridges to Stem Cells
Program, the UC Davis Institute for Regenerative Cures, CSU Sacramento Department of
Biological Sciences, and California Institute of Regenerative Medicine to study stem cell
biology. It is because of these institutions that I can participate alongside a community of
biological researchers and doctors striving to expand our understanding and capacity to promote
the foundations and practice of human health and well-being. For their profoundly encouraging
support and mentorship, I am immensely grateful to Drs. Min Zhao, Jan Nolta, Gerhard Bauer,
Jing Liu, and members of the Zhao lab, notably Drs. Junfeng Feng, Yao Hui Sun, and Lei Zhang.
From the CSUS family, I am indebted to Drs. Thomas Peavy, Thomas Landerholm, Christine
Kirvan, and Susan Lindgren for their guidance. And though hardly ever given the full credit they
deserve, I would like to express my deep gratitude to my family and friends who provide me the
security and peace of mind in knowing that I am loved and in their thoughts. To my mother,
father and sister, you’ve been right all along. And for all others who were involved in bringing
this project into fruition, thank you, especially Brian Fury and Gregorio Gutierrez, who I expect
to address as Drs in the near future. This work bears my name but it carries the work of all these
giants.
vii
TABLE OF CONTENTS
Page
Acknowledgements ...................................................................................................................... vii
List of Tables ..................................................................................................................................x
List of Figures ............................................................................................................................... xi
INTRODUCTION ..........................................................................................................................1
MATERIALS & METHODS .......................................................................................................13
Culturing H9-ESC Derived NSC for Migration Analysis.........................................................13
Chemical stimulation of NSC migration and motility ..............................................................14
Electric field (EF) stimulation of NSC migration and motility.................................................15
a) Chamber Preparation ............................................................................................................15
b) Application of Direct current EF Stimulation to hNSCs ......................................................17
Time lapse Recording of NSC Migration Behavior..................................................................19
Quantification of NSC Migration Parameters with Stimulation Modalities .............................19
Statistical Analysis of Migration Parameters ............................................................................19
RESULTS .....................................................................................................................................20
H9 hESC derived NSCs display characteristic designation and markers of NSCs...................20
hNSCs display motile but non-linear, highly tortuous random migration behavior ................21
Chemical stimulation alone shows no significant change in cell motility ................................23
Electrical stimulation significantly increases hNSC motility and directionality of migration..29
Chemical and electrical co-stimulation increases hNSC directedness but not motility
consistently ..............................................................................................................................34
viii
DISCUSSION ...............................................................................................................................39
Literature Cited .............................................................................................................................47
ix
LIST OF TABLES
Tables
Page
Table 1 Values of significance between NSC track speeds with chemical stimulation ................ 28
Table 2 Values of significance between NSC speeds and directedness with electrical stimulation.
......................................................................................................................................... 33
Table 3 Values of significance between NSC speed, directedness and distance when comparing
electrical stimulation with chemical-electrical co-stimulation. ....................................... 37
Table 4 Values of significance comparing net displacement of EF only to chemical-electrical costimulation.. ..................................................................................................................... 38
x
LIST OF FIGURES
Figures
Page
Figure 1 Mechanism generating electric field at wound site. ......................................................... 9
Figure 2 Construction of EF Chamber.. ........................................................................................ 16
Figure 3 Electrotaxis Experiment Set Up.. ................................................................................... 18
Figure 4 H9 NSC exhibit motile phenotype in absence of stimulation......................................... 21
Figure 5 H9 NSCs display an actively motile but non-linear random migration in a
heterogeneous population.. ............................................................................................. 23
Figure 6 hNSCs speed stable for several hours but inefficient due to tortuous random behavior 24
Figure 7 Chemical stimulation of growth factor-starved hNSCs with bFGF and extracellular
ATP shows no increase in chemokinetic migration.. ..................................................... 26
Figure 8 Chemical stimulation of growth factor-starved hNSCs shows no increase in
chemokinetic migration in EGF stimulation. ................................................................. 27
Figure 9 NSC motility is enhanced when exposed to electrical field. .......................................... 30
Figure 10 hNSC migration is less tortuous and more directed towards cathode when exposed to
electrical field. ................................................................................................................ 31
Figure 11 hNSC migration directedness is quantified showing greater directional migration
towards cathode when exposed to electrical field. ......................................................... 32
Figure 12 EGF combined with EF stimulation increases migration directionality but does not
consistently increase motility. ........................................................................................ 35
Figure 13 Trends shows greater net horizontal displacement in EGF combined with EF
stimulation. Greater cathodal migration compared to just EF alone. ............................. 36
xi
1
INTRODUCTION
Acute traumatic brain injury (TBI) is the leading cause of long term disability in adults
under 40, affecting 150 to 200 per million people globally (Fleminger and Ponsford, 2005). In
the US alone, an estimated 1.7 million medical cases were reported annually from 2002 to 2006,
resulting in 52,000 deaths (Faul, 2010). Cases vary widely and depending on the location and
extent of injury to the brain, any number of physiological functions, bodily controls or cognitive
functions could be permanently altered or lost. In many ways, TBI is an extremely expensive
condition. Aside from the cost of years of physical therapy or in-home care, is the cost of
permanent loss of full physical capacity and personal autonomy. But perhaps more devastating is
the loss of cognitive functions such as critical thinking, reasoning, memory, language, sensory
perception, and emotional intelligence, many functions we as a society consider to be
foundations of being uniquely human. As a corollary, many patients must endure the
psychological tolls of coping with long term disability and the changes to how they and society
now perceives them. Additionally, as a nation, the direct and indirect fiscal cost of TBI injuries
amounts to approximately 60 billion dollars in hospitalization, emergency treatments, post injury
care, therapy and loss of occupational productivity (Finkelstein, 2006). For patients with the
means to access resources, a multi-facetted treatment plan can be crafted involving a
combination of surgical intervention, routine physical/occupational therapy and psychological
counseling. Though a variety of these post-injury interventions are available, the recovery plan
largely remains one of a rehabilitative strategy to assist endogenous recovery and retraining
rather than a strategy of regeneration. Therefore, the long term outcome of such injuries to the
central nervous system depends in large part on the age, general health and the capacity for
2
neurogenesis and regeneration in the individual (Faul, 2010). Coincidentally, the highest rate of
TBI-related fatalities falls primarily in the elderly population (Faul, 2010). In children and young
adults, we see predominantly long-term or permanent physical and cognitive handicaps (Faul
2010). Therefore there is a pressing need to improve our current therapies to produce greater
functional outcomes and actual regenerative tissue repair following TBI.
In traumatic brain injury, immediately after trauma, an acute inflammatory response
recruits innate immune system components and astroglial cells to the lesion site. They
phagocytose cellular debris, migrate towards damaged tissue and quarantine it by creating a
barrier of glial cells (Richardson, 2010, Namas, 2009, Lo, 2010). The proliferation of astrocytes
at the injury site repopulates the lesion with non-neuronal cells and secretes inhibitory
proteoglycans, extracellular filaments and matrices that begin to form a glial scar around the
lesion. This precedes a secondary phase of pathologies that radiate outward into healthy tissue
causing diffuse demyelination, neuronal axon loss, excitotoxicity and neuronal cell death
(Richardson, 2010, Namas, 2009, Lo, 2010). In both phases, the operative pathology involves the
acute or delayed loss of mature neuronal networks and the upregulation of mechanisms which
inhibit neo-neurogenesis and engraftment.
However, in response to mechanical injury and ischemia, human and rodent brains
themselves can up-regulate endogenous adult neural stem cell activity (Cayre, 2009, Lo, 2010).
Neural stem cells (NSCs) which retain their multipotency into adulthood, reside in a few select
regions of the brain and play a role in homeostatic neural tissue maintenance. NSCs provide a
new source of neuronal and glial progenitor cells that can differentiate and replace lost neurons,
astrocytes and oligodendrocytes (Jin, 2010). In rodents, neuroblast cells endogenously proliferate
and migrate towards the anterior region of the brain to the olfactory bulb where they support the
3
high turnover rate for new interneurons. Likewise in animal models of stroke, neurodegenerative
disease and mechanical injury, the major stem cell niches of the brain, such as the subventricular
zone (SVZ), upregulates NSC proliferation and their egress out of the niche (Reekman, 2012,
Cayre, 2009, Lo 2010, Zhang, 2011). This paramedic response to injury is maintained over the
course of a lifetime, though notably this capacity diminishes significantly with age (Cayre, 2009,
Chen, 2011). Previous studies have reported incidences of neural precursor cell migration from
the cell niche over a year after insult, demonstrating the persistence of endogenous NSC
recruitment (Cayre, 2009).
Much of current translational neuroscience research focuses on characterizing and
optimizing the transplantation of stem cells in acute and progressive neurotrauma animal models.
This effort has produced many studies showing positive increases in functional recovery, making
a strong case for hNSC transplantation as a future cellular treatment (Tsuji, 2010, Shimada,
2011, Lo, 2010, Reekman, 2012). Though the exact mechanism is yet to be determined,
transplanted NSCs have been shown to survive in the host brain and migrate towards the site of
injury. Engrafted cells demonstrated both molecular and electrophysiological markers of
terminally differentiated cells (Tsuji, 2010, Shimada, 2011). Additionally, NSCs can modulate
the T-cell activation response and secrete neurotrophic growth factors that support cell survival
and neuroprotective functions (Lo, 2010, Reekman, 2012). Research in this field has also applied
the technology behind growing NSCs from embryonic stem cell (ESC) lines to create a source of
cells for research and potential clinical use (Reekman, 2012). Though the science is still far
from supporting a standard sustainable supply of clinical grade NSCs, such a resource would
eliminate the need for an allogeneic or autologous tissue harvest (Reekman, 2012). Several
4
animal trials have already reported successful integration of ESC derived cells following
intracranial transplantation. (Denham, 2012, Tsuji, 2010, Kriks, 2011)
Yet despite their regenerative capacity and trophic support, the endogenous recruitment
of NSCs is still insufficient to significantly regenerate lost tissue and recuperate damaged neural
functions. The inflammatory response and astrocyte hypertrophy creates an inhospitable
microenvironment for recruited precursor cells, reducing their survivability and migration into
the glial scar (Reekman, 2012, Cayre, 2009). These microenvironmental conditions that
attenuate the recruitment of endogenous stem cells likely are also responsible for the limited
engraftment and migration of exogenous stem cell transplants as well. Additionally, NSCs, such
as those produced from the SVZ, are mostly confined to migrating within a designated structure
or neural tracts, and thus their range of mobility is restricted barring a major disruption of its
structural integrity. It is estimated that only 20% of migrating NSCs survive and, in a study of
stroke model, only .02% of lost neurons were replaced by endogenous recruitment (Jin, 2007,
Cayre, 2009). With respect to the clinical timeline post injury, the majority of the stem cell
recruitment activity begins and peaks after injury onset but drops off shortly after (Lo, 2010).
Beyond this refractory phase, the level of cumulative damage from secondary pathologies
continues to increase over time such that beyond a certain threshold, intervention may no longer
be effective (Lo, 2010). Efficient and robust migration of cells during this refractory period is
therefore particularly important to mounting an effective intervention. Until there is an effective
method for efficiently and quickly guiding cells to the site of injury, poor rates of cell
engraftment and recruitment will remain an obstacle to functional NSC based regeneration.
5
One method to enhance NSC motility is stimulating with chemokines to direct
migration. What was found early in the discovery of NSCs is their endogenous trophism towards
glioma tumors (Cayre, 2009, Heese, 2005). Glioma cells release a myriad of growth factors and
cytokines that promote cell proliferation such as basic fibroblast growth factor and epidermal
growth factor (Heese, 2005, Chicoine and Silbergeld, 1997). However these were also found to
induced a chemotactic response and for many mitogens, a corresponding chemokinetic effect as
well (Heese, 2005, Chicoine and Silbergeld, 1997). In addition, extracellular ATP, which is a
released in a paracrine fashion by neuronal and glial cells, has also been found to have a trophic
and chemotactic effect on neural stem/progenitor cells (Grimm, 2010). These factors are also
known to keep NSCs undifferentiated and to maintain multipotency, a state which is normally
transient when outside of the stem cell niche. Because these chemokines NSCs, this study
investigates the contributions of basic fibroblast growth factor, epidermal growth factor and
extracellular adenosine triphosphate to increasing NSC motility.
Basic fibroblast growth factor, or bFGF-2, is one of 20 members in the fibroblast growth
factor (FGF) ligand family (Boilly, 2000). It is a standard component of embryonic and neural
stem cell culture for maintaining pluripotency (Reekmans, 2011) but it was previously found to
promote cell proliferation and have a chemotactic/chemokinetic effect on glioma cells and neural
stem cells in vitro (Yamamoto, 1997, Heese, 2005, Erlandsson, 2004). Systemic infusion of
bFGF and EGF after NSC transplantation shows greater migration towards lesion site
demonstrating its effect on motility in vivo as well (Cayre, 2009). Endogenously, it is produced
by neurons and astrocytes and is one of many neurotrophic factors releases following injury
(Mattson and Scheff, 1994). Mechanistically, bFGF binds and activates its corresponding FGF
receptor (Boilly, 2000) via phosphorylation from SH2 domain signal transducers. The receptor
6
activates the G-protein molecular switches Rac, RhoA and Rho kinase (which are critical to
adhesion and lamellipodia formation during migration (Abe, 2007)) and enzyme kinases such as
Src, Protein Kinase C and PI3-kinase (Boilly, 2000).
Epidermal growth factor (EGF) is similar to bFGF in several ways. EGF is also a
neurotrophic growth factor released from astrocytes in response to injury. It too has positive
effects on cell proliferation and motility (Reekman, 2011, Grimm, 2010, Mattson and Scheff,
1994). EGF is a small peptide growth factor that binds to EGFR (a member of the receptor
tyrosine kinase family of transmembrane receptors) (Boockvar, 2003, Liebmann2011). Similar
to other RTK type receptors, it can activate downstream effector enzymes such as Stat3,
Phospholipase C, the Akt pathway and the MAPK ERK pathway (Boockvar, 2003, Liebmann,
2011).
Compared to the previous chemokines, extracellular adenosine triphosphate ATP has
only been recently recognized as a cell signaling factor rather than as a metabolic product. The
extracellular release of nucleotides, such as ATP and ADP, are mediated by most cell types as
well as during cell death in a wound or stress scenario (Zimmerman, 2006, Siow, 2010,
Burnstock, 2011). Nucleotide ligands are detected by two classes of purinergic receptors, P2X
ionic channels and P2Y metabotrophic G-protein coupled receptors (Fischer, 2007, Ferrari,
2011). The family of receptors is composed of subsets of members with higher affinity to a
specific nucleotide (i.e. ATP vs. ADP vs. UTP) (Zimmerman, 2006, Burnstock , 2011). Different
classes of purinergic receptors trigger different signaling pathways regulate multiple cellular
functions such as proliferation, migration, apoptosis, differentiation and self-renewal. Not
surprisingly, specific subsets of purinergic receptor are found expressed only during the
undifferentiated stage of neural stem cells (Siow, 2010, Burnstock, 2011). Extracellular
7
nucleotide-mediated purinergic signaling plays a role in regulating the migration of progenitor
and stem cells (Cayre, 2009, Ferrari, 2011, Zimmerman, 2006). Specifically, ATP and ADP
activate several P2Y receptors that trigger intracellular pathways often associated with migration
of neural cells (Liu, 2008, Agrestia, 2005). These include the phosphorylation of Phospholipase
C, MAPK ERK signaling pathways, focal adhesion kinase and induction of temporary
intracellular Ca2+ spikes (Burnstock, 2010, Zimmerman, 2006, Grimm 2010). A recent study of
hMSCs also shows purinergic signaling 1) upregulates genes involved in cytoskeleton
reorganization while downregulating genes in cell adhesion and proliferation, and 2)
significantly increases spontaneous migratory behavior even in the absence of chemokines
(Ferrari, 2011). Extracellular ATP-activation can produce transitory Ca2+ influxes and promote
the migration of oligodendrocyte progenitor and differentiated cells (Siow, 2010, Burnstock,
2011, Cayre, 2009).
In addition to chemical cues, physical stimuli, specifically, electric fields (EFs), can also
direct cell division and migration direction, in some cases as an ‘overriding’ dominant cue
(Zhao, 2006, 2009, Forrester, 2006). This is interesting particularly because by nature and
function, the brain itself is an electric organ and NSCs theoretically exist within an environment
of endogenous electrical activity. To explain wound directed electrotaxis, an epithelial wound
models best illustrates a simplified scenario of wound healing in endogenous EF (Zhao, 2009,
Forrester, 2007, Figure1). The voltage potential behind electric fields originates from the ion
channels and transporters on the apical and basal sides of the membrane, mediating the
transmembrane ion concentration (Na+, K+, and Cl-). The apical side of the tissue typically
maintains a net opposite charge relative to the basolateral side, creating a transepithelial voltage
8
potential. This voltage potential can be measured in skin, corneal epithelium, respiratory,
gastrointestinal, urinary and retinal epithelial tissues as well (Zhao, 2009, Forrester, 2007).
Disruption of the cell layer (i.e. a wound) presents a breach in this charge separation, allowing
ions to migrate and circumvent this barrier creating an electrical current and the wound EF.
Endogenous wound EFs have been recorded in multiple systems including, human and rodent
skin and corneal wounds (Zhao, 2009). Its strength depends on wound size and follows a voltage
gradient from as high as 140mV along the wound edge down to 0mV towards the center of the
wound as measured in guinea pigs and human skin (Zhao, 2009, Forrester, 2007).
9
Transport of Ions
Na
+
Transepithelial
Potential
0mV
++
Wounding
--
-
Transport of Ions
Na+
Cl
-
+25~40m
V
Wound Electric
Fields
Transe
-
--
+
+
2
5uA/cm
Figure 1 Mechanism generating electric field at wound site. Epithelial model of
membrane potential shows segregation of positive and negatively charged ions by a
membrane barrier creating a voltage potential. Breach in the barrier (i.e. wound) creates
an electrical current in which cells may migrate (as illustrate by bold outlined cells). Cells
shown here migrate towards the cathode but different cell types migrate to different
poles. (Image created based on figure by Zhao (2009)
Cl-
+25~
10
Near the wound edge, cells in mitosis detect and divide along the EF vector and migrate along
available surfaces in the direction of the EF vector from the more positive ‘pole’ towards the
wound edge with an oppositely charged ‘pole’ (Zhao, 2009, Forrester, 2007). As the wound
remains open, the wound EF is maintained by intact peripheral and basolateral cells surrounding
the wound that preserve homeostatic ionic concentrations. This keeps the wound edge
differentially charged compared to the void of cells and voltage at the wound center until the
epithelial barrier is restored (Zhao, 2009, Forrester, 2007).
There are several pivotal studies that also recapitulate electrotaxis in vitro using
exogenous EFs. The overriding effect of an EF in vitro was most dramatically demonstrated in a
scratch wound assay where cells in unison can switch migration direction when an EF was
applied and then reversed. Collective electrotaxis occurred even away from the wound against
the direction of contact inhibition pressures and chemical gradients (Zhao, 2009). Electrotaxis
was also confirmed not to be a chemotaxis artifact when media cross flow experiments
demonstrated that in spite of consistent fluid flow to remove any confounding chemotactic
gradients created via electrophoresis, cells maintained a directed migration response towards one
electrical pole in the presence of an electrical stimulus (Song, 2007, Yao, 2011). In vitro, cells in
electrotaxis not only migrate directionally but in some cases, faster (Meng, 2010, Zhao, 2009).
This electrotactic behavior has been observed in several different cells types (including
keratinocytes, corneal and vascular endothelium and neurons) and in more recent studies with
stem cells and early progenitor cells as well (Arocena, 2010, Zhao, 2009, Meng, 2010, Yao,
2011, Zhang, 2011). Electrotaxis in an exogenous DC electric field has been observed in
cultured cell monolayer models, stratified tissue and cultured 3D environments (Zhao, 2009,
Zhang, 2011). Surprisingly, endogenous processes such as directional nerve sprouting, vessel
11
growth and neurogenesis respond to this force as well (Song, 2004, 2007, Zhao, 2009) and
possibly even prenatal development (Yao, 2011, Saunders, 2005). To date, it is not know how
cells detect EFs or if there is an EF ‘receptor’ however several intracellular pathways regulating
electrotaxis have been identified. Cells exhibit polarized intracellular gradients of actin fibers
and adhesion molecules towards the leading edge (Zhao, 2009). Similar to chemotaxis signaling
pathways, electrotaxis (of neutrophils, keratinocytes, NPCs and epithelial cells) shows polarized
intracellular gradients of activated MAPK signaling, Src, Pten and PI3K towards the leading
edge in parallel to the EF vector (Meng, 2010, Zhao, 2002, 2009). Interestingly, cells in EF also
demonstrate an asymmetrical concentration of membrane lipids, surface receptors, and
polarization of Golgi apparatus suggesting a biased presentation of decorated cell membrane and
ligand detection at the leading edge (Meng, 2010, Zhao, 2009). Exogenous EFs have also been
known not only to alter distribution but to modulate the expression of signal receptors such as
EGF receptors (Zhao, 2002). Additionally, NPCs and neural cells display increased intracellular
Ca2+ signaling as mediated through voltage-gated Ca2+ channels and endoplasmic reticulum
stores (Yao, 2011, Meng, 2010). All of these intracellular signals are represented during
chemotaxis and migration, verifying not only the molecular mechanisms behind electrotaxis but
also the potential signal crosstalk between electrical and chemical migration cues. It suggests
that there may be an additive benefit in improving directional migration due to convergent
intracellular signaling and transactivation of neighboring receptors.
During wound healing, it is likely that both neurotrophic chemical release and wound
electric field are present. NSCs migrating in this signaling milieu will likely be exposed to both
stimuli and could possibly be an endogenous component of their recruitment to the injury site. A
few possible convergent transduction pathways include MAP kinase ERK pathway, Src, PI3K
12
signaling pathways and an increase in intracellular Ca2+ waves (Cayre, 2009, Zhao, 2002, 2009,
Meng, 2010). It therefore begs the question if exogenous chemical and electrical co-stimulation
would improve NSC migration efficiency. Though research in this area is very limited, it
suggests an additive chemokinetic effect may be co-opted to improve the migration and
engraftment level of NSC transplants, thereby enhancing their regenerative and neuroprotective
functions. In addition, as a utility within the toolkit of stem cell-based biotechnology, the ability
to direct stem cell migration has considerable possibilities in future biomedical and basic
research applications.
In this study, we investigated migratory behavior of hNSCs in vitro in response to
exogenous chemical (bFGF, EGF and ATP) and electrical stimulation. Our studies suggest that
direct current electrical field significantly increases cell motility and directionality to a greater
degree than chemical stimulation alone. However using a co-stimulation modality enhances
directional migration but not due to additive motility.
13
MATERIALS & METHODS
Culturing H9-ESC Derived NSC for Migration Analysis
Human embryonic stem cell line (H9) derived neural stem cells (NSCs) were kindly provided by
Dr. Jing Liu (Institute of Regenerative Cures). Liquid N2 frozen cells are rapidly thawed at 37°C
before immediately resuspended in warm media to dilute the cytotoxic cryoprotectant, dimethyl
sulfoxide (DMSO). Cells are collected by pelleting via centrifugal force and resuspended again
with warm DMSO-free media. Multicellular clusters that shield ‘core’ cells from growth factor
signaling are likely to differentiate and produce impure cell populations. Therefore a completely
dissociated cell suspension is important to multipotent stem cell culture by allowing uniform
growth factor signaling necessary to maintain their phenotype and inhibit the propensity to
differentiate. Cells are seeded at a high density to promote paracrine signaling inducing cell
survival and proliferation. NSCs are seeded on .003% poly-L-Ornithine (Sigma) and 10ug/ml
laminin (Sigma) coated polystyrene tissue culture plates (BD Falcon) and cultured at 37°C, 5%
CO2, 100% humidity in Neurobasal Medium (Gibco) supplemented with 100uM MEM nonessential amino acids (Invitrogen), 2mM L-glutamine (Invitrogen), 1%
N2 Supplement
(Invitrogen), 1% penicillin/streptomycin (Invitrogen), .1% B27 Supplement (Gibco), 20ng/ml
basic fibroblast growth factor, bFGF (Invitrogen), and 10ng/ml epidermal growth factor, EGF (R
& D Systems). Media is changed every two days to remove waste products and replenish
nutrients and growth factors, allowing NSC cultures to expand four to fivefold into confluent cell
monolayers within 5-6 days. A confluent culture can then be passaged and divided into multiple
cultures to increase cell stocks. During passage, intercellular connections are digested with a
14
gentle proteolytic, collagenolytic solution, Accutase (Stem Cell Technologies). Cells are allowed
to recover for a minimum of two days post passage to restore surface receptor, remove dying
cells and allow entry into an active proliferation phase. Cells are used between the 4th and 10th
passage to prevent any major changes in morphology or migration that may occur due to
cumulative cell or DNA damage that may occur during passaging. At least 12hrs prior to
experimentation, NSC are starved in reduced growth factor culture media (5ng/ml bFGF and
2.5ng/ml EGF) to magnify effects of chemical agonists that may share convergent signaling
pathways with culture media growth factors.
Chemical stimulation of NSC migration and motility
3 x 104 cells are plated in laminin coated 24 well plates (Corning Costar) with 300-500ul of 25%
reduced growth factor media to adhere in 37°C, 5% CO2, 100% humidity culture conditions.
After one hour cell seeding period, cells are adapted to 37°C atmospheric conditions and CO2
independent media (Gibco) supplemented with 1% Penicillin/Streptomycin (Invitrogen), 2mM
L-glutamine (Invitrogen), here after referred to as ‘CO2 Media’. This media change is necessary
because exposure to atmospheric conditions alkalizes culture media beyond a tolerable range for
NSCs, therefore cells must be maintained in a buffering media that maintains stable neutral pH
for multiple hours. After CO2 adaptation, cells are observed for two hours before carefully
replacing media with 500ul CO2 media containing the chemical agonist. Chemical stimulants
used in the study include adenosine triphosphate disodium salt (Sigma) diluted in ddH20,
20ng/ml bFGF (Invitrogen) in .1% bovine serum albumin, BSA (Sigma), and 10ng/ml EGF (R &
D Systems) in 10mM acetic acid. Cell migration was recording for at least two hours following
agonist addition.
15
Electric field (EF) stimulation of NSC migration and motility
a) Chamber Preparation
Electrotaxis chamber preparation is detailed previously (Zhao et al., 2010, Song et al.2007).
[Illustrated in Figure 2 below] Briefly, two 10 x 22 mm chamber regions coated with laminin,
are flanked by 8 x 22mm No. 1.5 coverslips (VWR) secured by high vacuum grease (Dow
Corning). Media reservoirs are created with vacuum grease borders at the head and tail openings
of the chambers to control media flow. Following starvation period, NSCs are harvested with
Accutase and 3 x 10^4 cells in 200 to 300ul media is evenly seeded between cover slips.
Following cell adhesion to surface, media is changed to CO2 media. A 22 x 30 mm sterile
coverslip roof is secured over both cover slips with grease to seal the EF chamber, leaving a boxlike tunnel of uniform dimensions between reservoirs for media flow-thru. Reservoirs are filled
with approximately 3 ml CO2 Media and NSCs in EF chambers allowed to adapt to atmospheric
conditions at 37°C for one hour before time lapse recording of self-control treatment.
16
~.1mm thick
Figure 2 Construction of EF Chamber. Schematics of dish containing two electrotaxis chambers
based on design created by Song et. al (2007) that can be simultaneously imaged during the same
time period. Image on left shows the construction and dimensions of dual electric field (EF)
chamber with cells seeded in the chamber center. Media reservoirs on either side additional
media to buffer small temperature and pH changes in experiment and act as a conduit for direct
current to be applied across cells. Image on right shows magnified image of an EF chamber and
approximate placement of the agar bridges.
17
b) Application of Direct current EF Stimulation to hNSCs
EF application protocol is described in greater detail by the cited literature in references (Zhao,
Song et. al, 2007) [See Figure 3 for illustration of this system]. Briefly, direct current (DC)
power source (Consort EV265) is connected via leads to Ag/AgCl electrodes submerged in 1X
Steinberg’s solution. 1 to 1.5% Agar-Steinberg filled glass bridges contact Steinberg solution
which contains ionic salts to conduct current, and CO2 media reservoirs forming a completed
electrical circuit and EF vector through the seeded cells. Additionally EF voltage is significantly
reduced and dramatic changes in pH are buffered. Prescribed voltage strengths across chamber
are applied and monitored during recording. Two sets of agar bridges and Steinberg’s solution
beakers are required for experiments applying EF to both EF chambers simultaneously.
18
Figure 3 Electrotaxis Experiment Set Up. Stem cells seeded in the electrotaxis chamber are
exposed to DC current via agar bridges immersed in Steinberg solution. Current is conducted
from power source through silver electrodes and Steinberg solution for a complete circuit. The
double chamber design allows for simultaneous imaging of a no EF control group.
19
Time lapse Recording of NSC Migration Behavior
Cell migration is imaged with Zeiss Axiovert 40CFL inverted microscope and motorized stage
(BioPoint 2 Ludl Electronic Products) in a 37°C temperature controlled enclosure. Time-lapsed
images are recorded with Hamamatsu digital camera every 10mins and images are compiled into
videos using Simple PCI 5.0 imaging software (Hamamatsu).
Quantification of NSC Migration Parameters with Stimulation Modalities
Compiled image stacks are corrected for background tremble with Metamorph 7.0 imaging suite
(Molecular Devices) in order to minimize any augmentation of data due to shaking of the
motorized stage. Migration speed and distance for 50 to100 cells per treatment are calculated by
manual tracking of migrating cells with ImageJ analysis software (NIH, Wayne Rasband).
Quantification of migration parameters was performed with ImageJ 1.46 and Microsoft Office
Excel 2007. Track speed is treated as a ‘spontaneous’ speed, of a ratio of total distance travelled
in time interval divided by time interval. Migratory distance is measured as accumulated distance
(total distance travelled) and Euclidean distance (net geometric displacement from the start site).
Directedness of migration is measured as cosine of the angle between the migration path vs.
electric field vector.
Statistical Analysis of Migration Parameters
Data is compiled using Microsoft Office Excel 2007 and statistical significance was determined
with IBM SPSS Statistics 19 by with-in subject repeated measures ANOVAs and Bonferroni
confidence interval correction or paired and unpaired Student’s T-test with and significance for
both set at p< .05.
20
RESULTS
H9 hESC derived NSCs display characteristic designation and markers of NSCs
The current protocol for deriving NSCs from ESCs requires weeks of careful
manipulation of the extracellular signaling environment. Embryonic stem cells colonies are
detached from adherent culture and maintained in floating suspension to induce the formation of
an embyroid body, a sphere comprised of cells differentiating into the three primary germinal
cell layers. Reattachment of an embryoid body to an adherent culture surface signals cells to
further differentiate and recapitulate the process of neural tube development as seen by the
formation of a neural rosette. Columnar cells at the rosette center express the NSC markers,
nestin (an intermediate filament) and sex determining region Y - box2 (SOX-2) (a pluripotency
associated transcription factor). When extracted and cultured, H9-NSCs demonstrate the
characteristic bi/tripolar cell morphology (Figure4a) and demonstrate the capacity to form 3D
neurosphere-like aggregates (data not shown). In addition, they display cytoplasmic staining for
nestin and nuclear staining for SOX-2 (Fig 4b/c/d, photos provided by Dr. Jing Liu).
21
a)
10x Bright field Phase contrast
c)
Nestin
b
)
Merged
d
)
SOX2
Figure 4 H9 NSC exhibit motile phenotype in absence of stimulation. a) Cultured H9 hNSCs
starved to 25% of culture growth factors display bipolar and tripolar cell morphology
characteristic of neural stem cells. b) NSCs are cultured and passaged til higher
passages numbers (12th) before staining for nestin and SOX-2 expression. Merged
labelling shows localization of nestin and SOX-2 staining in relation to cell. c) Nestin
specific staining coincides with cytoplasmic region of cells. d) SOX-2 staining is
localized in nuclear compartment and is round coinciding with shape of nucleus.
22
hNSCs display motile but non-linear, highly tortuous random migration behavior
In vitro migration studies demonstrate fundamental migratory behaviors unique to
specific cell types. To observe basal migration behavior of hNSCs, cells were plated on a
laminin coated surface and maintained in a reduced growth factor, serum-free CO2 media as they
are observed in a timelapse digital video recording. The basal level of migration was measured
as a function of speed and distance travelled. In the absence of exogenous stimulation, NSCs are
highly motile (Figure 5). However the extent of cell motility is heterogeneous between cell
passages and even between individual cells within a population (Fig 6a). However when
averaging a significantly high cell count, a defined migration trend and a small standard error
can be achieved.
Cells can also migrate quite quickly, at its peak travelling 50.88 um/hr (+1.82 um/hr) but
slowing as time progresses (at 8 hrs 40.188um/hr + 1.77, p=.00015, d.f=172) (Figure 6b).
However track speed is a dynamic parameter, speed measurements changes significantly when
measured on an hourly basis, appearing even more stochastic so at shorter time points with
alternating higher and lower speeds. In order to establish a stable baseline in which to compare
treatments but still show overall trends, ‘spontaneous’ migration speeds are measured in 2hr
intervals. Cells also travelled a significant distance, nearly 4/10th of a millimeter (369.95um +
11.62) cumulatively over an eight hour period (Figure 5, 6c). However the actual distance
travelled away from the cell’s starting site is dramatically less (89.64um + 4.59) (Figure 6c),
demonstrating the migratory path is non-linear or tortuous and as seen in time lapsed recording,
sometimes circling around it’s start point (Figure 5). Additionally the directionality of cell
migration is low (.019 + .053) as expected due to the cell’s random migration in the absence of a
directional cue (Fig 5, 6d).
23
No Stimulation-2hrs
No Stimulation-6hrs
No Stimulation-4hrs
No Stimulation-8hrs
No Stimulation-6hrs
Circling tortuous migration pattern
Figure 5 H9 NSCs display an actively motile but non-linear random migration in a
heterogeneous population. Time-lapsed analysis of manually tracked cell migration over
2hr intervals displays distance and tortuosity of migration paths.
No Stimulation-8hrs
24
a)
b)
c)
d)
Figure 6 hNSCs speed stable for several hours but inefficient due to tortuous random behavior.
a) Cells migrating in absence of exogenous stimulation migrating for 8 hours. Wide distribution
of migration speeds of cells within a sample population but little change over time. b) Average
track speed of population peaking at 50.88um/hr (+ 1.82um/hr) not significantly different for
first six hours but decreasing after eighth hour (40.18um/hr + 1.77um/hr, df=2.290, F=14.519,
p=1.878 x 10-7). c) Cells migrate cumulatively after 8hrs on average 369.96 um (+ 11.62 um,
df=3, f=14.519, p=4.32 x 10-9) while net Total Euclidean displacement of cells after 8hrs is
89.64 um (+ 4.59 um) migrating a significant distance away from starting point (df = 2.208,
F=90.414, p=2.513 x 10-35). d) Directedness of migration paths is measured from 1
(completely parallel with horizontal axis going from left to right) to 0 (completely parallel with
vertical axis). Cells travelled in absence of stimulation with directedness value of 0.019 (+
0.053), due to randomness of migration, a wide range of sample values results in a small
average and larger standard error. Directedness did not significantly change over time (df =
2.23, F= 2.006, p=0.130.)
25
Chemical stimulation alone shows no significant change in cell motility
NSCs were starved of growth factors, to reduce the level of growth factor signaling
associated with culture conditions that may interfere with their effect in migration and sensitize
cells. In order to preserve NSC multipotency, growth factor starvation was shortened to 12 hours
rather than 24 hours typically found in other migration studies. In vitro chemotaxis studies
typically demonstrate an effect by looking for an increased occurrence of NSCs in the region of
the highest ligand concentration. However the majority of in vitro studies employ a transwell
experimental system thereby creating an artificial concentration gradient to study agonist guided
chemotaxis. To study the chemokinetic effect of bFGF, EGF and ATP, a uniform concentration
is presented following a two hour basal self-control migration period. When exposed to
concentrations previously reported to direct cell migration (Heese, 2005, Grimm, 2010), bFGF
(at 5ng/ml and 20ng/ml), EGF (at 1ng/ml, 5ng/ml and 20ng/ml) and ATP (at 1uM, 10uM,
100uM), hNSCs appear to have no significant positive increases in track speed (Figure 7 and 8).
In some cases, the track speed slightly decreases as with bFGF (Figure 7a) and 1 and 10uM ATP
(Figure 7b, Table 1). The self-control migration period for EGF stimulation could not be
recovered and is therefore not included however a larger p-value was noted compared to the
vehicle suggesting a more significant change in speed compared to the control (Figure 8, Table
1c). However in either case, the small degree of change may have little to no biological
significance with regards to motility.
26
b)
a)
Add Agonist
Agonist
d)
c)
b)
*
*
Add Agonist
Figure 7 Chemical stimulation of growth factor-starved hNSCs with bFGF and
extracellular ATP shows no increase in chemokinetic migration. Specifically a & c) hNSC
migration distance and b & d) speed during a 2hr self-control period before changing to
CO2 media with bFGF vehicle (.1% bovine serum solution), 5ng/ml and 20ng/ml bFGF or
ATP vehicle (double distilled water), 1uM, 10uM and 100uM ATP. Track speed slows
significantly in the 4nd hour for 5ng/ml and 20ng/m bFGF (Table 1a).
27
Figure 8 Chemical stimulation of growth factor-starved hNSCs shows no
increase in chemokinetic migration in EGF stimulation. hNSC migration
distance and speed for recorded for a 2hr self-control period before changing
to CO2 media with EGF vehicle (10mM acetic acid solution), 1ng/ml, 5ng/ml
and 20ng/ml EGF. No self control data is available to show. Therefore
comparisons between the 2hr and 4hr timepoints were made and against
values from the vehicle treatment
28
Table 1 Values of significance between NSC track speeds with chemical stimulation
a) P-values from repeat measure ANOVA test of cell motility following exposure to bFGF
vehicle (.1% bovine serum solution), 5ng/ml and 20ng/ml bFGF b) ATP vehicle (double
distilled water), 1uM, 10uM and 100uM ATP, c) EGF vehicle (10mM acetic acid solution),
1ng/ml, 5ng/ml and 20ng/ml EGF via repeated measures ANOVA with Bonferroni confidence
correction
a)
Group
Period
N
df
F
bFGF vehicle
5ng/ml bFGF
5ng/ml bFGF
20ng/ml bFGF
20ng/ml bFGF
SC vs. 4hr
SC vs. 4hr
143
192
192
230
230
2
1.711
1.846
-
2.472
4.494
8.375
-
Group
Period
N
df
F
ATP Vehicle
ATP Vehicle
ATP Vehicle
ATP Vehicle
1uM ATP
1uM ATP
1uM ATP
1uM ATP
10uM ATP
10uM ATP
10uM ATP
10uM ATP
100uM ATP
SC vs. 2hr
SC vs. 4hr
2hr vs. 4hr
SC vs. 2hr
SC vs. 4hr
2hr vs. 4hr
SC vs. 2hr
SC vs. 4hr
2hr vs. 4hr
-
154
154
154
154
164
164
164
164
169
169
169
169
170
1.645
1.734
1.799
2
4.068
9.375
4.708
2.185
Group
Period
N
df
F
EGF Vehicle
1ng/ml EGF
5ng/ml EGF
20ng/ml EGF
-
246
229
306
229
1
1
1
1
26.22
16.48
60.36
61.23
Sig (p)
values
0.039
8.65 x 10-4
Sig. (p) with-in
subject effects
0.0862
0.016
4.068 x 10-4
-
Sig (p)
values
0.058
0.093
1
1.08 x 10-4
0.009
1
0.004
0.239
0.837
-
Sig. (p) with-in
subject effects
0.025
2.526 x 10-4
0.012
0.114
Sig (p)
values
-
Sig. (p) with-in
subject effects
6.143 x 10-7
6.74 x 10-5
1.21 x 10-13
1.88 x 10-13
b)
c)
29
Electrical stimulation significantly increases hNSC motility and directionality of migration
Despite cell-to-cell differences in migration behavior, most cells exposed to electric field
responded collectively (Fig 9a). On average, hNSCs in EF migrated a longer accumulated
distance compared to the no EF growth factor starved control (Figure 9b, Table 2) and a greater
net Euclidean distance from it’s starting point (Figure 9c). Comparing the two EF strength
conditions, though their accumulated distances are similar, cells in 300mV/mm migrates a longer
Euclidean distance compared to100mV/mm EF. This suggests the migration paths of cells in a
lower voltage strength involves more circling or non-linear migration behavior. The track speed
is also significantly higher compared to the no EF control (Figure 9d, Table 2). Most strikingly,
the migration of most cells is uniformally directed towards the negitive cathodal pole (Figure
10). Compared to the control treatment which shows cell disperal radiating outward in all
directions, cells in EF shows migration paths significantly skewed along a horizontal axis
(Figure 10). Thus the directedness value of cells in EF is also significantly higher compared to
no EF and directedness is corresponds to voltage strength (Figure 11a, Table 2). This is also
reflected in the net x-axis displacement of the migration tracks of cells in EF (Figure 11b).
However, it was also observed in the EF stimulation groups, compared to the control group
showed the CO2 media alkalized after recording overnight, that the CO2 media had alkalized
sooner and was acclerated with a higher voltage strength (data not shown). This pH change
required a media change in the EF chamber, which was also conducted for the control group,
and an adjustment of the EF field approximately every two hours. A delay in this change usually
resulted in mass cell death (data not shown).
30
b)
a)
No Electric Field – 4hrs
Apply EF
c)
Apply EF
300mV/mm Electric Field – 4hrs
+
_
d)
*
*
Apply EF
Figure 9 NSC motility is enhanced when exposed to electrical field. a) Baseline hNSC
migration is observed in self-control for two hours before applying 100, 300mV/mm or no EF
for four hours EF stimulation. Linear migration to cathode is seen. b) Quantification of total
accumulated migration distance of average cell with no EF, 100mV/mm and 300mV/mm EF.
c) Quantification of net Euclidean distance before and during four hours of EF stimulation
shows more linear migration path compared to no EF or a lower voltage. d) Quantification of
‘spontaneous’ track speed during EF stimulation. Motility dramatically increases with
application of EF.
31
a)
Self-Control
2hr EF
4hr EF
No Electric Field
b)
Self-Control
300mV/mm EF
2hr EF
+
4hr EF
-
+
-
Figure 10 hNSC migration is less tortuous and more directed towards cathode when
exposed to electrical field. a) Cells in the absence of EFs show no change even
distribution of cells migrating outward in all directions. b) Conversely, cells in electrical
field travel predominantly travel towards the right in the direction of the cathodal pole.
Plot graphs of cell migration show migration path of each treatment which quantify the
number of cells with net displacement to the left (in black, towards the anode) or the right
(in red, towards the cathode). Cross represents center of mass for all cell migration
32
a)
Apply EF
b)
Apply EF
Figure 11 hNSC migration directedness is quantified showing greater directional migration
towards cathode when exposed to electrical field. a) Quantification of directedness of cell
migration with no EF, 100mV/mm, 300mV/mm which is most directional at 300mV/mm EF.
b) Net migration distance of hNSCs travelled along x-axis direction with and without EF
stimulation.
33
Table 2 Values of significance between NSC speeds and directedness with electrical
stimulation a) comparing ‘spontaneous’ track speed during no EF stimulation, 100mV/mm and
300mV/mm EF stimulation and b) quantification of directedness of cell migration with no EF,
100mV/mm, 300mV/mm via repeated measures ANOVA with Bonferroni confidence
correction.
a)
Group
Period
N
df
F
Sig. (p)
No EF Ctrl
100mV EF
100mV EF
100mV EF
100mV EF
300mV EF
300mV EF
300mV EF
300mV EF
SC vs. 2hr
SC vs. 4hr
2hr vs. 4hr
SC vs. 2hr
SC vs. 4hr
2hr vs. 4hr
218
128
128
128
128
190
190
190
190
1.92
2
1.42
59.17
Group
Period
N
df
F
Sig. (p)
No EF Ctrl
100mV EF
100mV EF
100mV EF
100mV EF
300mV EF
300mV EF
300mV EF
300mV EF
SC vs. 2hr
SC vs. 4hr
2hr vs. 4hr
SC vs. 2hr
SC vs. 4hr
2hr vs. 4hr
202
128
128
128
128
190
190
190
190
1.92
1.35
2
-
1.42
35.49
93.11
-
1.05 x 10-6
1.021x 10-9
0.001
8.09 x 10-13
7.40 x 10-16
4.38 x 10-4
Sig. (p) with-in
subject effects
0.24
7.99 x 10-22
1.11 x 10-18
2.53 x 10-10
0.0019
1.80
1.42 x 10-30
93.11
-25
7.20 x 10
5.56 x 10-20
1
b)
Sig. (p) with-in
subject effects
0.242
1.65 x 10-10
1.16 x 10-16
-
34
Chemical and electrical co-stimulation increases hNSC directedness but not motility consistently
In the wound healing microenvironment, both chemical and electrical signaling occur
simultaneously and in all likelihood, act synergistically with each other. To investigate the effect
of this co-stimulation, we applied a 200mV/mm EF to cells in media containing 20ng/ml EGF.
200mV/mm EF strength was applied to attenuate some of the physical changes to the media as a
result of a higher voltage. Due to the limited availability of data and large deviation in values
between experiments, all results are presented to demonstrate the level of variation between
these experiments but also to highlight the trend seen in both. The basal cell migration speed for
this set of experiments is near or higher than peak migration speeds seen in the control assay
(Figure 12a). So from the beginning, the motility of these cells are higher than previous
experiment’s cultures. However because the self-control migration speeds between the EF
stimulation only vs. chemical and EF combined stimulation condition were similar, the trend
they exhibit appears valid (Table 3). With the application of 200mV/mm EF, the migration speed
jumps significantly for during a two hour period but eventually decreases back to near baseline
(Figure 12a). Similar to the EF stimulation experiments, the directedness of migration also
increases significantly and the migration is more linear along the x-axis towards the negative
cathodal pole compared to its self control baseline (Figure 12b, 13, Table 3). Compared to the
chemical-electrical co-stimulation modality, directedness is increased and the migration path of
most cells becomes more linear after the application of EF (Figure 12b, 13). However there is no
overall significant increase in the accumulated distance travelled.
35
a)
Apply EF
Apply EF
Trial 1
Trial 2
b)
Apply EF
Apply EF
Trial 1
Trial 2
Figure 12 EGF combined with EF stimulation increases migration directionality but does not
consistently increase motility. a) Track speed of NSCs during EF only or EF with 20ng/ml EGF
stimulation found to migrate at similar speeds. b) Migration Directedness of migration in
200mV EF vs. 200mVEF +20ng/ml agonist. Directedness in the co-stimulation modality is
higher compared to EF alone.
36
Apply EF
Trial 1
Apply EF
Trial 2
Figure 13 Trends shows greater net horizontal displacement in EGF combined with EF
stimulation. Greater cathodal migration compared to just EF alone.
37
Table 3 Values of significance between NSC speed, directedness and distance when comparing
electrical stimulation with chemical-electrical co-stimulation. P-values of significance of a) track
speed of NSCs during EF only or EF with 20ng/ml EGF stimulation, b) migration directedness
of migration in 200mV EF vs. 200mV/mm EF +20ng/ml agonist in co-stimulation modality
compared to EF only. P-values are derived via paired and unpaired Student’s T-test.
a)
Trial
Group
Period
N
1
1
1
1
1
1
2
2
2
2
2
2
200mV/mm EF only
200mV/mm EF only
200mV/mm EF only
20ng/ml + 200mV/mm EF
20ng/ml + 200mV/mm EF
20ng/ml + 200mV/mm EF
200mV/mm EF only
200mV/mm EF only
200mV/mm EF only
20ng/ml + 200mV/mm EF
20ng/ml + 200mV/mm EF
20ng/ml + 200mV/mm EF
SC
SC vs. 2hr
SC vs. 4hr
SC
SC vs. 2hr
SC vs. 4hr
SC
SC vs. 2hr
SC vs. 4hr
SC
SC vs. 2hr
SC vs. 4hr
87
87
87
57
57
57
80
80
80
77
77
77
Trial
Group
Period
N
1
1
1
1
1
1
2
2
2
2
2
2
200mV/mm EF only
200mV/mm EF only
200mV/mm EF only
20ng/ml + 200mV/mm EF
20ng/ml + 200mV/mm EF
20ng/ml + 200mV/mm EF
200mV/mm EF only
200mV/mm EF only
200mV/mm EF only
20ng/ml + 200mV/mm EF
20ng/ml + 200mV/mm EF
20ng/ml + 200mV/mm EF
SC
SC vs. 2hr
SC vs. 4hr
SC
SC vs. 2hr
SC vs. 4hr
SC
SC vs. 2hr
SC vs. 4hr
SC
SC vs. 2hr
SC vs. 4hr
87
87
87
57
57
57
80
80
80
77
77
77
Sig. (p) with
Self Ctrl
1.38 x 10-8
.991
4.85 x 10-9
.044
.237
2.44 x 10-6
.022
.407
Sig. (p) between
subject - EF only
.774
.0629
.0923
.826
.051
.0209
Sig. (p) with
Self Ctrl
3.65 x 10-15
2.371x10-15
2.16 x 10 -10
2.8 x 10-11
5.46 x 10-6
4.36 x 10-5
1.99 x 10-9
1.76 x 10-8
Sig. (p) between
subject - EF only
.57
.15
.021
.509
.0035
.0047
b)
38
Table 4 Values of significance comparing net displacement of EF only to chemical-electrical costimulation. P-values of significance compares Trials 1 and 2, 200mV EF vs. 20ng/ml EGF and
200mV/mm EF, derived via paired and unpaired Student’s T-test.
Trial
Group
Period
N
1
1
1
1
1
1
2
2
2
2
2
2
200mV/mm EF only
200mV/mm EF only
200mV/mm EF only
20ng/ml + 200mV/mm EF
20ng/ml + 200mV/mm EF
20ng/ml + 200mV/mm EF
200mV/mm EF only
200mV/mm EF only
200mV/mm EF only
20ng/ml + 200mV/mm EF
20ng/ml + 200mV/mm EF
20ng/ml + 200mV/mm EF
SC
SC vs. 2hr
SC vs. 4hr
SC
SC vs. 2hr
SC vs. 4hr
SC
SC vs. 2hr
SC vs. 4hr
SC
SC vs. 2hr
SC vs. 4hr
87
87
87
57
57
57
80
80
80
77
77
77
Sig. (p) with
Self Ctrl
2.20 x 10-18
3.24 x 10-18
2.10 x 10-15
1.02 x 10-15
2.32 x 10-9
1.02 x 10-7
2.6 x 10-18
3.8 x 10-16
Sig. (p) between
subject - EF only
.245
.031
.002
.36
.0002
.0001
39
DISCUSSION
Where endogenous tissue repair efforts fail to induce a functional recovery following
neurotrauma, NSC transplantation shows potential for better functional recovery and tissue
repair. Adult neural stem cells are widely thought to serve as a reservoir for new neural cell
generation with an ambulatory function during neurotrauma. However a major obstacle in stem
cell transplants is the efficient migration of cells to the site of injury to allow engraftment at
injury site. Though many studies exploit chemotactic gradients or methods of sensitizing
chemokine receptors to direct cell migration, fewer have looked at the use of physical cues to
direct cell migration. Even fewer have applied an electric and chemokinetic co-stimulation
condition. The relevance of such a study is that though electrical fields alone have been shown to
direct cell migration, in reality, it is unlikely that chemical or electrical migration cues act
independently in an in vivo wound healing setting. Our studies examined how NSC migration
behavior is affected by chemical, electric and combined stimulation modalities.
Without any exogenous stimulation, our observations confirmed the intrinsic motility of
hNSCs. Our in vitro migration model is a biologically minimal environment because it does not
provide an optimal network of adhesion substrates, surfaces, interstitial fluid flow, 3D
intercellular cues or microenvironmental signaling found in an in vivo environment. Yet NSCs
can migrate substantially, nearly 4/10th of a millimeter in just six hours on a permissive substrate,
laminin, which is commonly associated with blood vessels and astrocytes. Additionally these
cells were previously starved of bFGF and EGF growth factor signaling suggesting that few
signaling factors are required in order to see significant NSC migration. There are also a limited
number of studies that measure time-lapsed migration parameters such as distance and speed.
40
Compared to published rodent in vivo measurements, the hNSC migration speed found here is
similar to those in vivo which fluctuates between 20-70um/hr and displays a similar stochastic
migration behavior of alternating fast and slow migration periods (Bovetti, 2007). It suggests
that our in vitro migration model allows for similar migration patterns as those seen in vivo.
Our experiments suggest that growth factor starved NSCs when exposed to a chemical
motogen in the absence of a concentration gradient, exhibits no significant positive chemokinetic
change in baseline migration parameters such as migration speed or distance. In actuality, EGF
signaling appeared to slow cell motility slightly. The chemical agonists used in this study are
known to guide cell migration via chemotactic gradients (Heese, 2005, Zimmerman, 2006).
However to study a chemokinetic effect, no concentration gradient was used and instead, a
uniform ligand concentration was applied and expected to trigger greater, though random, cell
motility. Additionally because these cytokines signal convergent intracellular signaling pathways
such as the ERK and MAPK pathways also triggered during electrotaxis, a positive migration
change was expected. Within the context of these studies, our negative results could point to
several important ideas:
Firstly, growth factor starvation beforehand may have diverted the cellular program from
migration towards proliferation. Pre-experimental starvation is used to distinguish the lingering
effects of cell culture growth factor signaling from the change in motility during
experimentation. However, the cytokines we tested for chemokinetic properties are also involved
in maintaining cell survival and proliferation as it is the case that most mitogens also have an
effect on motility. They even require activation of similar intracellular factors such as protein
kinase C (PKC) and ERK2 in bFGF (Boilly, 2000), EGF and ATP signaling. For pleiotrophic
factors such as bFGF, the status of cell health and cell number can direct the receptor signal
41
towards regulatory pathways associated with proliferation (Boilly, 2000). It is possible that a
pre-starvation period may change hNSC in vitro migration to a ‘nutrient seeking’ motile
behavior. But upon exposure to neurotropic levels of environmental mitogens (such as a media
change containing a high concentration of agonists), the cells may slow or stop migrating. Thus
the activation of the same surface receptor may signal more cells to stop and divide rather than
migrate, lowering the average migration speed for the sample population.
Secondly, activation of ligand receptors is very tightly regulated. Upon ligand binding
and activation of surface receptors, they are quickly endocytosed and degraded or uncoupled
from their G-protein signaling intermediate (Bennett, 2011). The presence of a continuous
uniform chemical signal may induce extended receptor desensitization; ‘communicating’ to the
cell it has arrived in a region sufficient to support survival. This is one regulatory mechanism
that prevents continuous stimulation by preventing excess presentation of active surface
receptors on the cell membrane (Bennett. 2011). Likewise, there may be extracellular methods of
regulation that prevent excessive signaling by degrading ligands in the extracellular space as is
the case for extracellular ATP and ectonucleotidases (Zimmerman, 2006). Conversely, when
looking at cellular mechanisms behind cancer metastasis, more than 40 oncogenes encoding
tyrosine kinase surface receptors for chemokines such as bFGF, EGF and hepatocyte growth
factor (HGF) are found to be overexpressed (Mareel & Leroy, 2003). This would appear to be an
effective strategy to dodge the complications of motogen/mitogen receptor regulation however
this overlooks the possibility of a corrective role behind signal desensitization in the correct
guidance of cells in situ. As demonstrated by Minina et al. (2007), when CXCR4 receptor for a
potent chemokine, stromal derived factor-1 (SDF-1) is constitutively expressed on zebrafish
primordial germ cells, PGCs show ectopic chemotaxis towards multiple physiologically
42
incorrect sources of SDF-1. Therefore it is possible that our results reflect extended receptor
desensitization or ligand degradation resulting in the cell maintaining a homeostatic level of
intracellular signaling contributing to migration and therefore little to no change in cell motility.
In contrast, direct current EF simulation dramatically increases cell motility and
migration directionality compared to a chemical stimulant alone. It has the surprising effect of
not only directing cell migration en mass towards the same general direction, but also increasing
migration speed and net distance travelled. Previous studies of cell migration in electric fields
have demonstrated that the phenomenon does not stem from an artificial electrophoretic ligand
concentration gradient (Babona-Pilipos et. al, 2011) though it doesn’t necessarily rule out that
this may contribute in part to the increase in directedness or motility. By exposing cells to an
electric field, many intracellular signaling intermediates and surface receptors are concentrated
in one direction. This includes Rac, Rho kinase and other cytoskeleton regulators that determine
form the leading edge. It also establishes a persistent cell polarity in which secretory vesicles and
actin polymers can be concentration. Conversely, exposure to a uniform chemical ligand
concentration presents a uniform global activation of surface receptors. Instead of a chemotactic
gradient which provides a directional cue to establish a differential level of signaling from the
leading vs. the lagging edge, signal cascades are fired from all directions, preventing the
formation of a dominant polarity in one direction. It is analogous to a cell which travels in one
committed direction compared to a cell receiving signals from all ends and constantly changing
directions. A similar behavior was reported by Pankov et.al. (2005) that reported suppression of
Rac activity lead to formation of single dominant protrusions that lead cell migration.
Conversely, global Rac activation, likely by uniform growth factor or purinergic receptor
induces formation of many peripheral protrusions competing for cell polarity (Pankov, 2005).
43
However when combining these two modalities, we observed the directionality of NSC
electrotaxis is significantly higher. Previous studies demonstrated electric fields can polarize
surface membrane receptor (Zhao, 2009). One current hypothetical model demonstrates the
alignment of chemical receptors along the direction of the electric field which may explain the
observance of increased directedness of migration, and it is therefore possible that activation of a
concentration of receptors on one side of the cell may increase directedness when combined (Wu
and Lin, 2011). There were not sufficient repetitions to validate if this trend is a validated
phenomenon the direction of results appears consistent. Unfortunately there is no data for bFGF
and ATP-EF stimulation conditions due to restrictions of time and resources. Though because of
the similar role these cytokines play, a similar trend in increasing migration directionality
suggests a common intracellular role for motility factors in electric field. Possibly in
upregulating mechanisms that promote cell polarity, amplifying intracellular signaling in
response to cell’s detection of an electric field or actin cytoskeleton dynamics that are distinct
from those involved in motility.
One of the most important lessons learned from this of this project was how dynamic
pure cell population can be even under consistent culture conditions and media. The NSCs
provided in this study were generously donated from Dr. Jing Liu, who oversees the
differentiation of H9 embryonic stem cells to NSCs. Many pilot and data gathering experiments,
both successful and otherwise, were completed with these cells but they were by no means a
consistent cell line. Stable cell or tumor cell lines proliferate quickly, produce cells that respond
consistently between passages events and quite resistant to changes environment. NSCs on the
other hand are by experience particularly susceptible to in vitro culture conditions. To maintain
44
NSC cell state in vitro, cultures must be passaged periodically to prevent overgrowth and cell
senescence. The process of passage is usually quite traumatic to NSCs, killing at least 10% with
each pass. For each progressive pass, the individual surviving cells can over time accumulate cell
damage that changes proliferation and migration behavior. Based on experience, cells at later
passages tend to have slightly different morphologies and are more difficult to passage additional
times. To avoid the possibility of differentiation or genetic mutations due to repeated cell
damage, the number of passages acceptable for experiments was kept within a narrow range.
NSCs were received, at the earliest, four passage events after differentiation and all experiments
were performed with cells between 6-10 passages. Cell motility and proliferation patterns appear
to change between passage numbers and this is evident in the experimental results by the varying
baseline speeds between experiments.
Likewise the timing of the culture period from the day of passage to the day of
experimentation was also relatively rigid. To promote survival and proliferation following a
passage, cells are usually plated at a high density to allow paracrine and autocrine survival
signaling. This is usually effective at recovering cells during culturing phase. The consequence
of plating cells at high density is the time between passages to 100% culture confluence is
relatively short. Whereas a stable cell line can be plated at very low densities, less than 10%
confluence, and still be expected to grow for several days before reaching confluence. It required
a balance act of maintaining cell cultures long enough to grow enough cells for future
experiments but not long enough to collect cells when they are beyond the log phase of their
growth curve. The timing of this phase also varied as passage numbers accumulated. Minor
changes in the passage event were also found to change migration behavior during time lapse
recording. During culture period, cells can recover post passage over several days and by and
45
negative effects from the passage are not seen. As little as 30 seconds longer during digestion
period or cell dissociation by pipetting could dramatically change cell motility. With less time to
recover from disturbances, the difference between cultures becomes more apparent.
Another important technical aspect learned during this project is the importance of
physical environmental factors on cells when the scale becomes increasingly small. During an
experiment, cell density is intentionally low for purposes of individual cell quantification to
minimize intercellular interactions that introduce another variable factor. Unfortunately, higher
culture densities that provide greater cell-cell contact promote survivability and tolerance to
environmental changes such as media temperature and flow. Larger media volumes used in
culture are also better at buffering physical changes. Whereas in an experiment changing media
during an experiment it also has the unwanted side effect of creating fluid flow of only a few
milliliters of liquid through an opening .1mm2 big. These were similar concerns regarding pH
change, physical vibrations and even light exposure. Though the scale of our model does not
deal in microliter volumes, the technical difficulties of this model mimics those found when
working with microfluidic devices.
Technical complications aside, the relevance of the aforementioned project rests on the
implications of the findings to an in vivo setting. Though the in vitro experimental system is
biologically minimal, it is also somewhat of a privileged condition for cell migration. It lacks the
same cytotoxic conditions, inflammatory response and recruitment of innate immune system
components to the injured brain and is therefore is less stressful compared to an in vivo postinjury environment. Additionally, migration of cells on an unobstructed 2D surface is
significantly different compared to 3D tissue network of extracellular matrix and interconnected
46
cells. However in vitro migration assays can demonstrate the basic cellular response using a
simplified stimulation construct. It is known that electrical and chemical gradients and cues have
general concurrent roles in directing mass cell migration behind tissue regeneration and also
early embryonic development. In this manner, we can investigate the response level that can be
attributed to each type of stimuli. In our studies here, the preliminary data presented suggests
that a chemical and electrical co-stimulation method may enhance the efficiency of EF guided
NSC migration. Though we did not see an additive or sustained increase in motility, it suggests
that once optimized, EGF plays a role in increasing directedness. Conversely, it suggests that
with an optimized administration of EGF, a lower EF strength can be applied in order to achieve
the same level of directed migration. This model also only represents two classes of stimuli in a
process that involves many other factors affecting tissue repair/formation forces. Therefore, the
ability to identify differences in response to migration cues in vitro compared to in vivo is can
help tease out other mitigating factors and the magnitude of its effect on the organ’s overall
ability to direct cell migration and ultimately tissue formation and repair. On a larger scale, it
highlights the importance of biophysical forces in wound healing biology that is only beginning
to develop acceptance in translational medicine. Furthermore, it represents a largely unexplored
area of biomedical science yet to be explored.
47
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