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.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS November 10, 2011.
Small direct current (DC) electric fields (EFs) guide neurite growth and migration of rodent neural stem cells (NSCs). However, this could be species dependent. Therefore, 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 migration toward the cathode. Reversal of the field polarity reversed migration of hNSCs. The galvanotactic/electrotactic response was both time and voltage dependent. The migration directedness and distance to the cathode 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 blockage of CXCR4 did not affect the electrotaxis of hNSCs. We conclude that hNSCs respond to a small EF by directional 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 diseases. STEM CELLS 2012; 30:349–355.
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 transplanted/endogenous neural stem cells (NSCs). When NSCs were transplanted into the rat adult hippocampus, they incorporated 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. Unfortunately, very few NSCs survive if directly transplanted to the site of damage . Therefore, it is more plausible to transplant 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 proliferation over terminally differentiated neurons. Significant beneficial 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 to an EF cannot be simply deduced from previous publications, because the guidance effect of EFs for cell migration and neurite growth has significant interspecies difference and is cell type dependent. For example, neurites from Xenopus neurons grow remarkably well toward the cathode, 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 pluripotent stem cells (hiPSCs) and hESCs showed completely different 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 strategies 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 clinically 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 into neurons and astrocytes. For neuron differentiation, hNSCs were cultured in neurobasal medium supplemented with B27, brain-derived neurotrophic factor (BDNF), ascorbic 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 amino acid (NEAA). NSC population was expanded in neural induction 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 video-microscopy.
Cells were pretreated with either Y27632, a Rho-kinase (ROCK) inhibitor (0, 10, 25 μM), or C-X-C chemokine receptor type 4 (CXCR4) antagonist AMD3100 (0, 5, 25 μg/ml; from Sigma, St. Louis, MO, http://www.sigma.com) for 0.5 hour in a CO2 incubator before the treatment of electrotaxis experiment.
We use the following parameters [28, 34, 37]: (a) directedness = cosine (θ), where θ 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 +1. A value close to 0 represents random migration. The cosine (θ) will range from −1 to +1, and an average of cosine (θ) yields the directedness value for a population of cells, giving an objective quantification of the direction of cell migration. (b) Track speed (μm/hour): accumulated migrated distance in 1 hour. (c) Displacement speed (μm/hour): the straight line distance from the starting point to the final position of cell in 1 hour. (d) X-axis distance (μm): 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.
Data are expressed as mean ± SEM. Statistical analysis was performed using SPSS software with unpaired, two-tailed Student's t-test (time- and strength-dependent electrotaxis experiment), or analysis of variance (ANOVA) (Y27632, AMD3100 experiments). p was set at .05 for rejecting null hypotheses.
RESULTS AND DISCUSSION
To confirm NSC features of the derived cells, we showed differentiation sequence of H9 ESCs, embryoid body formation, and rosette isolation as previously reported . Immunofluorescence 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 β-III-tubulin-positive neurons and GFAP-positive astrocytes (Fig. 1).
We first determined the response of hNSCs to an EF. Different 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 different 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 electrical signal to facilitate stem cell therapy, it is therefore important 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).
To determine the threshold voltage for EF-directed migration, we subjected the cells to EFs of different strength. The electrotaxis of hNSCs is time and voltage dependent with a threshold of 16 mV/mm or below. Cells showed gradually increased cathodal 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 survival . 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 mesenchymal 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 directedness 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-1α (chemokine (C-X-C motif) ligand 12 (CXCL12) or SDF-1α), 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 labeled 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 elucidating 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 difficult if not impossible to control in vivo. Compared to these biochemical 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 complicated 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 experiments using electric stimulation together with other guidance molecules (BDNF, nerve growth factor, and netrins) and ultimately in vivo experiments will be needed to elucidate interaction between the electrical and biochemical signals. Electric stimulation in combination with other cues (growth factors, cytokines, etc.) is likely to lead to a more effective guidance strategy for hNSCs.
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 supported 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 a fellowship from the Shriners of Northern California.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
M.Z. has research funding/contracted research with CIRM.