TISSUE-SPECIFIC STEM CELLS
Brief Report: Guided Migration of Neural Stem Cells Derived from
Human Embryonic Stem Cells by an Electric Field
JUN-FENG FENG,a,b,cJING LIU,a,dXIU-ZHEN ZHANG,a,bLEI ZHANG,a,bJI-YAO JIANG,cJAN NOLTA,a,dMIN ZHAOa,b,e
aInstitute for Regenerative Cures,bDepartment of Dermatology,dStem Cells Program, Department of Internal
Medicine, andeDepartment of Ophthalmology, University of California Davis School of Medicine, California,
USA;cDepartment of Neurosurgery, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai,
People’s Republic of China;b;d;c
Key Words. Human neural stem cells•Directional cell migration•Electric field•Electrotaxis•Rho-kinase•C-X-C chemokine receptor
Small direct current (DC) electric fields (EFs) guide neu-
rite growth and migration of rodent neural stem cells
(NSCs). However, this could be species dependent. There-
fore, it is critical to investigate how human NSCs (hNSCs)
respond to EF before any possible clinical attempt. Aiming
to characterize the EF-stimulated and guided migration of
hNSCs, we derived hNSCs from a well-established human
embryonic stem cell line H9. Small applied DC EFs, as
low as 16 mV/mm, induced significant directional migra-
tion toward the cathode. Reversal of the field polarity
reversed migration of hNSCs. The galvanotactic/electrotac-
tic response was both time and voltage dependent. The
increased with the increase of field strength. (Rho-kinase)
inhibitor Y27632 is used to enhance viability of stem cells
and has previously been reported to inhibit EF-guided
directional migration in induced pluripotent stem cells and
neurons. However, its presence did not significantly affect
the directionality of hNSC migration in an EF. Cytokine
receptor [C-X-C chemokine receptor type 4 (CXCR4)] is
important for chemotaxis of NSCs in the brain. The block-
age of CXCR4 did not affect the electrotaxis of hNSCs.
We conclude that hNSCs respond to a small EF by direc-
tional migration. Applied EFs could potentially be further
exploited to guide hNSCs to injured sites in the central
nervous system to improve the outcome of various dis-
eases. STEM CELLS 2012;30:349–355
Disclosure of potential conflicts of interest is found at the end of this article.
Stem cells must migrate directionally to diseased or damaged
tissues to repair and to regenerate. Limited understanding
exists for the mechanisms guiding the migration of trans-
planted/endogenous neural stem cells (NSCs). When NSCs
were transplanted into the rat adult hippocampus, they incor-
porated into the upper blade [1, 2]. Many signaling molecules
have been suggested to guide the migration [3–5]. Damaged
brain tissue may signal to recruit transplanted embryonic stem
cells (ESCs) to damaged regions, even from the left caudal to
the right caudal and left frontal . Some types of damage
may need focal delivery of replacement cells, while more
widespread damage or damage of less-accessible parts of the
brain may require long-range dispersal of NSCs. Unfortu-
nately, very few NSCs survive if directly transplanted to the
site of damage . Therefore, it is more plausible to trans-
plant NSCs to the region adjacent to the damage and then
induce them to migrate to the damage. Endogenous NSCs
may be recruited to the damaged brain areas, but only small
portion of the newly produced NSCs are able to do so [7–9].
No clinically effective technique is currently available to
guide migration of human NSCs (hNSCs). Guiding migration of
hNSCs has direct clinical relevance. NSCs for clinical use must
be human. Using hNSCs minimizes tumorigenesis which may
be a drawback of using human ESCs (hESCs) , and hNSCs
have the advantage of ample supply, better survival, and prolif-
eration over terminally differentiated neurons. Significant benefi-
cial effects of transplanting hNSCs have been demonstrated in
animal models of stroke [11–13], Parkinson’s disease [14, 15],
spinal cord injury [16–19], traumatic brain injury [20, 21], and
brain tumor (as an effective delivery system) [22, 23].
Direct current (DC) electric field (EF) is an effective cue
to guide neurite growth and migration of neurons and other
types of cells [24–30]. Rodent NSCs migrate directionally in
an EF [26, 27, 30]. Unfortunately, how hNSCs would respond
toanEF cannotbe simplydeduced fromprevious
Author contributions: J.-F. F.: conception and design, collection and assembly of data, data analysis and interpretation, and manuscript
writing; J.L.: provision of study material, collection and assembly of data, and data analysis and interpretation; X.-Z. Z.: collection and
assembly of data; L.Z.: provision of study material and collection and assembly of data; J.-Y. J.: provision of study material; J.N.:
financial support, provision of study material, and final approval of manuscript; M.Z.: conception and design, financial support,
provision of study material, data analysis and interpretation, manuscript writing, and final approval of manuscript.
Correspondence: Min Zhao, M.D., Ph.D., Institute for Regenerative Cures, 2921 Stockton Boulevard, University of California Davis,
Sacramento, California 95817, USA. Telephone: 1-916-703-9381; Fax: 1-916-703-9384; e-mail: email@example.com
16, 2011; accepted for publication October 24, 2011; first published online in STEM CELLS EXPRESS November 10, 2011. V
Press 1066-5099/2012/$30.00/0 doi: 10.1002/stem.779
STEM CELLS 2012;30:349–355 www.StemCells.com
publications, because the guidance effect of EFs for cell
migration and neurite growth has significant interspecies dif-
ference and is cell type dependent. For example, neurites
from Xenopus neurons grow remarkably well toward the cath-
ode, those from rat neurons grow perpendicular in an EF, and
neurons from zebra fish do not respond to an EF at all [24,
31–33]. Our own investigation using human induced pluripo-
tent stem cells (hiPSCs) and hESCs showed completely differ-
ent electrotaxis. hiPSCs migrated to the anode, while hESCs
migrate to the cathode . Those findings from rodents and
from different human stem cells cannot be simply transferred
to human cells and to hNSCs derived from H9 ESCs.
Therefore, it is important to test whether hNSCs migrate
directionally in an EF. In an effort to develop practical strat-
egies to guide migration of more differentiated cells, we
derived hNSCs from a well-characterized hESC line H9 and
determined the response to applied EFs. Human NSCs are a
cell type of clinical potential for use in brain trauma, stroke,
and neurodegenerative diseases. Their responses are thus clin-
ically relevant and form an initial valuable and necessary step
before further evaluation in vivo.
MATERIALS AND METHODS
Derivation of NSCs from H9 ESCs
The multipotency of the derived hNSCs was confirmed by the
differentiation intoneurons andastrocytes. Forneuron
on feeder cells. (B): Embryoid bodies were formed in suspension culture. (C): Numerous clusters of columnar cells formed rosettes, after attach-
ment of the embryoid bodies for 1 week. (D): Rosettes were positively labeled with anti-SOX-1 and Nestin antibodies. (E): The isolated rosettes
were dissociated into single human NSCs after treatment with accutase. NSCs were cultured as monolayer adherent cells. (F): Immunofluores-
cence analysis showed that NSCs derived were positive for the neuroepithelial markers, Sox-1 and Nestin. (G): NSCs can be induced to differen-
tiate into b-III-tubulin-positive neurons. (H): NSCs can be induced to differentiate into GFAP-positive astrocytes. Scale bar ¼ 100 lm.
Abbreviations: GFAP, glial fibrillary acidic protein; SOX-1, sex determining region Y-box 1.
Characterization of neural stem cells (NSCs) derived from human embryonic stem cells (hESCs). (A): hESCs (line H9) were cultured
Electrically Guided hNSC Migration
differentiation, hNSCs were cultured in neurobasal medium supple-
mented with B27, brain-derived neurotrophic factor (BDNF), ascor-
bic acid, glial cell-derived neurotrophic factor (GDNF), and cyclic-
Adenosine monophosphate (AMP). For astrocyte differentiation,
hNSCs were cultured in neurobasal medium supplemented with 1%
B27, 1% N-2 supplement, 1 mM L-glutamine, and 1% non-essential
random direction when cultured without an EF. The white lines represent tracks of cell migration with the cell positioned at the end of migration at 1
hour. See Supporting Information Video 1. (B): hNSCs showed robust cathodal migration in an EF (300 mV/mm, for 1 hour). See Supporting Informa-
tion Video 2. (C): Migration trajectories of hNSCs for 1 hour in EFs with the starting point set at the origin. The unit of the axes is in microns. (D):
Directedness values of hNSCs in an EF of different field strength at 1 hour. The average cosine increased with field strength. (E, F): Reversed migra-
tions of the same hNSCs followed the reversal of the field polarity. The tracks of cell migration before (E) and after (F) the field polarity was reversed.
The unit of the axes is in microns. See also Supporting Information Video 3. (G, H): Reversal of migration direction indicated by the directedness
value (G) and X-axis distance (H) of hNSCs migration in an EF before and after polarity reversal. Data were analyzed based on the setting of 0-minute
as the start position for the first 80 minutes and 80-minute as the start position for the EF reversed 80 minutes. EF ¼ 250 mV/mm. Scale bar ¼ 100
lm. *, p < .05; **, p < .01 when compared to that of cells cultured in control conditions without an EF. Abbreviations: EF, electric field.
An applied direct current (DC) EF-directed migration of human neural stem cells (hNSCs) toward the cathode. (A): hNSCs migrated in
Feng, Liu, Zhang et al.
amino acid (NEAA). NSC population was expanded in neural induc-
tion medium plus 0.1% B27 and 10 ng/ml epidermal growth factor
(EGF) on poly-L-ornithine/laminin-coated dishes.
Details were previously reported [35–37]. Cells were seeded in
an electrotactic chamber coated with laminin, in CO2-independent
medium (Invitrogen, Carlsbad, CA, http://www.invitrogen.com/)
plus 1 mM L-glutamine for 0.5–2 hours before the electrotaxis
study. Cell migration was recorded using time-lapse digital
Cells were pretreated with either Y27632, a Rho-kinase (ROCK)
inhibitor (0, 10, 25 lM), or C-X-C chemokine receptor type 4
(CXCR4) antagonist AMD3100 (0, 5, 25 lg/ml; from Sigma, St.
Louis, MO, http://www.sigma.com) for 0.5 hour in a CO2incuba-
tor before the treatment of electrotaxis experiment.
We use the following parameters [28, 34, 37]: (a) directedness ¼
cosine (y), where y is the angle between the EF vector and a
straight line connecting the start and end position of a cell. A cell
moving directly along the field lines toward the cathode (to the
right) would have a directedness of þ1. A value close to 0 repre-
sents random migration. The cosine (y) will range from ?1 to
þ1, and an average of cosine (y) yields the directedness value for
a population of cells, giving an objective quantification of the
direction of cell migration. (b) Track speed (lm/hour): accumu-
lated migrated distance in 1 hour. (c) Displacement speed (lm/
hour): the straight line distance from the starting point to the final
position of cell in 1 hour. (d) X-axis distance (lm): the distance
which is projected on the X-axis (parallel to the EF direction)
from the starting point to the final position of cell’s migration.
Cells Markers and Staining
Cells were labeled with rabbit anti-human Sox1 (1:500, Millipore,
Billerica, MA, http://www.millipore.com [AB15766]); mouse anti-
human Nestin (1:500, R&D, Minneapolis, MN, http://www.
rndsystems.com/ [MAB1259]); rabbit anti-human glial fibrillary
acidic protein (GFAP) (1:1000, Millipore, Billerica, MA, http://
www.millipore.com [AB5804]); rabbit anti-TuJ1 (1:500, Abcam, San
Francisco, CA, http://www.abcam.com/ [ab24629]); or rabbit anti-
human CXCR4 (1:200, Thermo Fisher Scientific, Waltham, MA,
(Alexa Fluor 488 and 594 nm, respectively; Invitrogen, Carlsbad,
CA, http://www.invitrogen.com/) were applied for 1 hour. Cells were
mounted with VECTASHIELD mounting medium with 406-diami-
nino-2-phenylindole (DAPI) (Vector laboratories Inc., Burlingame,
Data are expressed as mean 6 SEM. Statistical analysis was per-
formed using SPSS software with unpaired, two-tailed Student’s
t-test (time- and strength-dependent electrotaxis experiment), or
analysis of variance (ANOVA) (Y27632, AMD3100 experi-
ments). p was set at .05 for rejecting null hypotheses.
RESULTS AND DISCUSSION
To confirm NSC features of the derived cells, we showed dif-
ferentiation sequence of H9 ESCs, embryoid body formation,
and rosette isolation as previously reported . Immunofluo-
rescence staining showed that columnar cells inside rosettes
were positive for neuroepithelial markers, Sox-1 and Nestin.
The derived NSCs continued to express those markers. After
weeks of directed differentiation, NSCs gave rise to b-III-tubu-
lin-positive neurons and GFAP-positive astrocytes (Fig. 1).
cosine) represented cumulative directional translocation measured at 10-minute intervals for 1 hour at the four field strengths. (B): X-axis distance
toward the cathode was measured at 10-minute intervals for1 hour. (C, D): The track speed and displacement speed of hNSCs for 1 hour. *, p <
.05; **, p < .01 when compared to that in no EF at 1 hour. Abbreviations: EF, electric field.
Voltage dependence and time dependence of the electrotaxis of human neural stem cells (hNSCs). (A): Directedness values (average
Electrically Guided hNSC Migration
We first determined the response of hNSCs to an EF. Differ-
ent types of cells, or even the same type of cells from different
species, responded remarkably differently to EFs. Robinson and
Cormie  made a detailed comparison of the responses of dif-
ferent neurons to EFs. One striking difference is that neurites
from Xenopus neurons showed directional growth in a very small
EF of approximately 8 mV/mm, while neurites from Zebrafish
neurons completely ignored the presence of an EF as high as
100 mV/mm, although the growth of neurites was the same [31,
32, 39, 40]. However, neurons from rodents did not respond to
applied EFs, or the neurites were orientated perpendicular to the
field direction, neither toward the cathode nor the anode [33, 39].
Neuron-like cells differentiated from PC12 cells orientated the
neurites toward the anode . Studies suggested that rodent
neural stem/progenitor cells migrate to the cathode in an EF [26,
27, 30]. To develop techniques to guide hNSCs exploiting elec-
trical signal to facilitate stem cell therapy, it is therefore impor-
tant to determine how NSCs of human origin respond to EFs.
In an EF, hNSCs migrated directionally to the cathode. Reversal
of the field polarity reversed the migration direction (Fig. 2).
tion trajectories of hNSCs with the starting point set at the origin. The axes are in microns. (B): Y27632 (10, 25 lM) had no significant effect on
directedness value of EF-directed migration. (C): Y27632 treatment decreased the track speed when no EF exposure or at low field strength (100
mV/mm) but had no effect on that of hNSCs at 300 mV/mm. D: hNSCs were positively stained for CXCR4. (E): Migration trajectories of hNSCs
after AMD3100 treatment. The axes are in microns. (F): AMD3100 (5, 25 lg/ml) did not inhibit the migration directedness of hNSCs in EF. (G):
No significant effect of AMD3100 on the track speed of hNSC migration with or without EF. Scale bar ¼ 100 lm. **, p < .01 when compared to
that in no drug treatment. Abbreviations: CXCR4, C-X-C chemokine receptor type 4; DAPI, 406-diamindino-2-phenylindole; EF, electric field.
Y27632 inhibiting ROCK and AMD3100 blocking CXCR4 did not affect electrotaxis of human neural stem cells (hNSCs). (A): Migra-
Feng, Liu, Zhang et al.
To determine the threshold voltage for EF-directed migration,
we subjected the cells to EFs of different strength. The electro-
taxis of hNSCs is time and voltage dependent with a threshold
of 16 mV/mm or below. Cells showed gradually increased catho-
dal migration with higher field strength (Figs. 2C, 2D, 3A, 3B).
The directedness value increased with EF strength. Additionally,
an EF of 300 mV/mm significantly increased cell track speed
and displacement speed (Fig. 3C, 3D).
We next examined the effects of Y27632 on EF-guided
migration of hNSCs. The compound Y27632 is used in stem
cell transplantation and passaging to promote stem cell sur-
vival . Y27632 inhibits the Rho A effectors ROCK 1 and
2. Y27632 treatment significantly decreased the track speed
when no EF or low EF stimulated, while did not affect the
directional migration of hNSCs in an EF (Fig. 4A–4C).
ROCK inhibition enhances post-thaw viability of human mes-
enchymal stem cells (hMSCs) and hESCs [43, 44] and helps
survival of transplanted ESC-derived NSCs . It may also
regulate neural differentiation [45, 46]. Inhibition of ROCK
using Y27632 significantly affected electrotaxis of human
iPSCs and rat hippocampus neurons [28, 34, 47]. The direct-
edness value of EF-directed migration of hNSCs, however,
was not sensitive to the Y27632 treatment. We finally tested
whether the well-studied chemotaxis pathway through CXCR4
is involved in the electrotaxis of hNSCs. CXCR4 is the primary
receptor for stromal derived factor-1a (chemokine (C-X-C
motif) ligand 12 (CXCL12) or SDF-1a), a potent chemokine for
stem cell migration. CXCR4 is a key molecule in chemotaxis of
many types of stem cells and regulates migration of NSCs
derived from ESCs [5, 48]. Evidence suggests that migration of
NSCs toward a tumor bed or to the ischemic sites in the brain
is also regulated by CXCR4 [49, 50]. CXCR4 is positively la-
beled in the derived hNSCs (Fig. 4D). Its antagonist AMD3100
had no significant effect on the directional migration or on the
track speed of hNSCs with or without EF exposure (Fig. 4E–
4G). These results showed that the guidance effect of DC EFs
is different from that of chemotaxis for hNSCs. There is a small
possibility that Y27632 and AMD3100 may have off target
effects. Further molecular experiments will be needed for eluci-
dating the exact signaling mechanisms.
EF has some unique properties and could be a technique
that compliments other therapies. Several potential methods to
direct migration of transplanted stem cells have been explored,
including enhancement on chemotaxis of stem cells through gene
manipulation of chemokines and their receptors such as SDF-1/
CXCR-4, cytokine pretreatment, and extracellular matrix breaking
down [48, 51–55]. For example, induced expression of CXCR4
in MSCs significantly increased homing of the cells to the site of
infarcted tissue in the heart . However, biochemical guidance
cues may be difficult to manipulate. There are very complicated
chemical gradients existing in vivo. Those coexisting directional
cues in vivo may not only be a confounding factor but also have
less predictable or controllable effects on stem cells to home to
injured sites or diseased tissues. Thus, chemical gradients are dif-
ficult if not impossible to control in vivo. Compared to these bio-
chemical methods, application of an EF has the advantages of
easy control of direction, magnitude, immediate application and
withdraws, with no chemical residuals. Application of EFs has
flexibility of varying strength, time, direction and space location,
almost adjustable at will. An applied EF might act on the compli-
cated chemical gradients in vivo. It is not known whether this
interaction may cause even more confounding effects in guidance
of hNSCs or may unify the guidance effects. Our in vitro results
suggest that the guidance effects of EFs on hNSCs appear to be
insensitive to ROCK inhibitor Y27632, which is a widely used
agent to help maintain stem cells. The SDF-1/CXCR-4 signaling
pathway, which is important for stem cell migration, does not
have significant effects on electrotaxis of hNSCs. Further experi-
ments using electric stimulation together with other guidance
molecules (BDNF, nerve growth factor, and netrins) and ulti-
mately in vivo experiments will be needed to elucidate interac-
tion between the electrical and biochemical signals. Electric stim-
ulation in combination with other cues (growth factors, cytokines,
etc.) is likely to lead to a more effective guidance strategy for
In summary, a small EF (16 mV/mm) is an effective cue to
guide migration of hNSCs. The guidance effect is different
from undifferentiated iPSCs which appeared to depend on Rho/
ROCK signaling and also different from chemotaxis through
CXCR4 pathway. Electric stimulation may offer a practical
approach to facilitate therapies using hNSCs in brain injury,
where guided cell migration and integration are needed.
We thank Dr. Lin Cao and other members from the Zhao and
Nolta laboratories for assistance. This work was supported by
grants from the California Institute of Regenerative Medicine
RB1-01417 (to M.Z.) and TR1-01257 (to J.N.). M.Z. is also sup-
ported by NIH 1R01EY019101, NSF MCB-0951199, and UC
Davis Dermatology Developmental Fund, and in part by the
Research to Prevent Blindness, Inc. J.N. is also supported by the
NIH (5P30AG010129, 5RC1AG036022-02, and 2P51RR000169-
49). J.F.F. is supported by NSFC (30901543). J.L. is supported by
DISCLOSURE OF POTENTIAL
CONFLICTS OF INTEREST
M.Z. has research funding/contracted research with CIRM.
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