Introduction

Neural precursors have remarkable migratory capacities, which play important roles during CNS development and which underlie cell transplantation therapies in animal models of neurodegenerative disease. Neural stem cell activity is found initially in the apical progenitor cells of the neural tube. As the neural epithelium thickens during early embryogenesis, the apical progenitors (radial glia) elongate and span the ventricular (apical) to pial (basal) surfaces of the CNS. The radial glia are the neural stem cells during embryonic neurogenesis and these cells may produce intermediate (basal) progenitors and neurons that migrate radially, away from the subventricular zone (Noctor et al. 2008). Most radial glia terminally differentiate or apoptose towards the end of CNS development, but some neurogenic progenitors persist into adult life. In the course of adult neurogenesis, these neural stem cells can still be found in the subventricular zone of the cortex (at least) and subgranular zone of the dentate gyrus. Subventricular cortical stem cells give rise to neuroblasts, which will then undergo a remarkable process of migration anteriorly across the brain, along a structure named the rostral migratory stream, to finally reach the olfactory bulb (Ghashghaei et al. 2007). These migratory events rely on diverse types of directional cues for precise targeting to the appropriate regions. For instance, neural stem cells have been observed to migrate radially across the cortex along the extended processes of other cells (Noctor et al. 2008), whereas neuroblasts in the rostral migratory stream respond to combinations of, among others, Slit-mediated chemorepulsive signals and netrin-1, GDNF- and BDNF-mediated chemoattractant signals (Chiaramello et al. 2007).

NSCs can be grown in vitro in large quantities, particularly via the formation of cell aggregates known as neurospheres from cortical, hippocampal or spinal cord explants, and can then be transplanted. Both in vitro and in vivo, neurosphere-derived cells show great capacity for differentiation into a wide variety of glial and neuronal cells types, consistent with a stem cell origin and indicating clinical potential for CNS repair. Transplanted NSCs have shown extensive migratory behaviors in several experimental models of brain and spinal cord pathology (Lindvall and Kokaia 2006), and they have also been demonstrated to have remarkable tropism towards brain tumors (Aboody et al. 2000). Transplanted NSCs also respond to different types of guidance cues to reach their targets. Recently, NSCs have been shown to display a strong directional migratory response when exposed to electric fields (EFs) both in vitro and in the environment of an ex vivo brain slice model (Arocena et al. 2010; Meng et al. 2011). EFs have been shown to be strong directional cues for a wide variety of cell types in vitro (McCaig et al. 2005), and their ability to control NSC migration could have potential clinical applications. This review will focus on the directional cues that guide NSC migration in the context of brain repair therapies and brain tumor targeting, and in the use of EFs to control cell migration, particularly NSC migration.

Directional Cues for Neural Stem Cell Migration in Brain Repair Therapies

Chemotactic signals are among the main cues that guide NSCs and more committed neural precursors during development, and this chemotactic response is mirrored when NSCs are transplanted in animal models of brain injury. For instance, in a mouse model of stroke, it was shown that in the cerebral hemisphere where ischemic injury is induced the cytokine SDF-1 is markedly up-regulated, and that NSCs transplanted to the contralateral hemisphere migrated towards high SDF-1 areas in the injured hemisphere (Imitola et al. 2004). NSCs expressed the SDF-1 receptor, CXCR4, and exposure to a chemical gradient of SDF-1 in vitro led to a marked increase in transmigration in a Boyden chamber. Moreover, when NSCs cultured as neurospheres were co-cultured with ischemic brain explants, NSCs migration out of neurospheres towards explants was remarkably stimulated compared to control explants, but this increase was abrogated by treatment with a blocking antibody against the SDF-1 receptor. SDF-1 is up-regulated by astrocytes and endothelial cells that are activated by the inflammatory milieu of the ischemic brain. As an inflammatory signature is common in different brain pathologies, gradients of cytokines produced in this process could become major directional cues for transplanted NSCs, contributing to the remarkable homing of NSCs to injured brain areas. A recent study has provided additional support for this concept, by showing that directed migration of NSCs transplanted into the dentate gyrus of mice with kainic acid-induced seizures is dependent on the cytokine CXCL12 (Hartman et al. 2010). Also, in vitro assays further confirm that many other cytokines, such as VEGF, PDGF-BB, RANTES and M-CSF, are chemoattractants for NSCs (Zhang et al. 2003; Schmidt et al. 2009).

Besides chemotactic signals, the topography of the brain environment could also be a source of directional cues for NSCs. For example, endogenous neural progenitors have been shown to move along blood vessels in the course of their migration towards damaged areas in brain slices from a mouse model of stroke (Kojima et al. 2010). In vitro, NSCs are responsive to engineered topographic cues, for example aligning in parallel to fibers with diameters ranging from 0.3 to 1.5 μm (Yang et al. 2005). In vivo, topographic cues could cooperate with other directional signals to help migrating NSCs maintain their course over long distances, enabling their migration to areas of brain pathology.

The extracellular matrix plays critical roles in cell migration, and there is evidence that its interactions with NSCs can modulate the directedness of their migration in the injured brain. In a mouse model of ischemia where neurons in the CA1 region of the hippocampus are selectively lost, a marked difference in directed migration of two neural stem cell clones after hippocampal transplant was observed, with one of the clones being much more effective at repopulating areas of neuronal death (Prestoz et al. 2001). The same difference in migratory performance was observed in assays in vitro with various extra­cellular matrix substrates, but it was abolished when interactions between these substrates and integrins were inhibited. As both clones expressed similar levels of several integrins, it was suggested that variations in integrin signaling could underlie their different migratory capabilities. It is therefore conceivable that interactions between the extracellular matrix and NSCs could act in combination with other guidance cues to promote directed NSC migration in vivo.

Directional Cues for Neural Stem Cell Migration Towards Brain Tumors

The phenomenon of NSC tropism towards brain tumors was originally described for intracranial glioma (Aboody et al. 2000), but has since been extended to medulloblastoma, neuroblastoma and melanoma brain metastases (Kim 2011). Directed migration towards tumors is robust, as NSCs transplanted intracranially at distant sites from the tumor mass or delivered intravenously were able to reach it and, remarkably, they were also observed to closely follow small groups of tumor cells migrating away from the main tumor mass (Aboody et al. 2000). As in the case of NSC migration to injured brain areas, the large amount and variety of cytokines produced in tumor areas constitutes a fundamental source of guidance cues for NSCs. In vitro, NSCs show strong chemotactic responses to cytokines produced by glioma cell lines, particularly VEGF and HGF (Kendall et al. 2008). The cytokine SDF-1 also plays an important role in glioma tropism, as NSCs migrate in vivo towards areas of the tumor with high levels of this cytokine, in particular the tumor border and hypoxic regions within the tumor (Zhao et al. 2008). Hypoxia leads to upregulation of SDF-1 and VEGF in glioma cell lines, whereas reactive astrocytes that accumulate at the tumor border might be another source of SDF-1 (Zhao et al. 2008). Therefore, a variety of diffusible signals produced by tumor cells, either normally or in response to hypoxia, and also by cells from the tumor stroma, can be exploited by NSCs as guidance cues, which contributes to the persistent tropism they display towards brain tumors.

Tumors also produce abundant extracellular matrix, which can profoundly alter the microenvironment surrounding tumor cells. Gliomas remodel the pre-existing extracellular matrix, synthesizing in particular large amounts of vitronectin, fibronectin and tenascin. All of these substrates promote NSC migration and, importantly, they induce high levels of NSC transmigration when they are used to coat the underside of a Boyden chamber filter (Ziu et al. 2006), showing that NSCs are capable of haptotaxis -the directed migration of cells in gradients of substratum-bound ligands- in response to extracellular matrix produced by gliomas. It is therefore conceivable that the local enrichment in certain adhesive molecules in the brain tumor microenvironment could operate as an additional directional cue for NSCs, cooperating with other guidance signals to strengthen their long distance migration towards the tumor mass.

Electric Fields as Directional Cues

EFs, defined as gradients of voltage across space, are vectors, and can orient charged particles, as can be observed in the classic images of the patterns formed by charged threads placed in EFs. Already by the end of the nineteenth century it was known that single-celled protists could migrate parallel to an EF vector. This directed migration phenomenon was termed galvanotaxis, or electro­taxis. Since then, many studies have reported the electrotactic behavior of a wide variety of cell types, from amoeboid single cells of Dictyostelium, to vertebrate cells such as keratinocytes, fibroblasts and neutrophils (Erickson and Nuccitelli 1984; Zhao et al. 2006). Different cell types have been observed to migrate either towards the cathode (the negative pole) or towards the anode (the positive pole). For instance, Dictyostelium, keratinocytes, and neutrophils normally migrate towards the cathode (Zhao et al. 2006), whereas Schwann cells and retinal pigment epithelial cells move towards the anode (Mckasson et al. 2008; Gamboa et al. 2010). These results show that the final orientation of the electrotactic response is not uniquely determined by the EF, but also by how different cell types interact with it.

When cells are exposed to an EF, charge movement is generated on the outer surface of the cells, where Na+ and other cations accumulate around negatively charged sugar moieties of transmembrane proteins and glycolipids. Cations will drag along water, generating an electro-osmotic flow, which has been shown to asymmetrically redistribute membrane proteins to the cathode-facing side of cells (McLaughlin and Poo 1981).

Cathodal redistribution of membrane receptors is an early event after EF exposure. For example, in keratinocytes, the epidermal growth factor (EGF) receptor localized preferentially on the cathodal cell membrane as early as 5 min after the beginning of EF exposure (Fang et al. 1999), and fast cathodal redistribution of the EGF receptor has also been demonstrated in corneal epithelial cells (Zhao et al. 1999). Inhibition of the EGF receptor reduced its cathodal accumulation and subsequent cell electrotaxis, suggesting that asymmetric signaling from this receptor is required for electrotaxis (Fang et al. 1999).

The phosphatidylinositol-3-OH kinase (PI3-K) pathway has also featured as an important component of the signaling events that lead to electrotaxis. Keratinocytes, fibroblasts and neutrophils from mice deficient in the p110γ catalytic subunit of PI3-K showed markedly impaired electrotactic responses (Zhao et al. 2006). Conversely, cells from mice deficient in the PI3-K antagonist PTEN display enhanced electrotaxis. Also, the same study showed that, similar to what occurs in chemotaxis, the PI3-K product PIP3 accumulates at the leading edge of cells undergoing electrotaxis.

A model of electrotactic migration has been proposed in which cathodal accumulation of activated receptors such as the EGF receptor would initiate localized signaling events such as activation of the PI3-K pathway, which in turn would stimulate protrusive activity, transforming the cathodal side of cells into the leading edge and directing cell migration towards the cathode (McCaig et al. 2005). Intracellular calcium can participate in sensing electric gradients; for instance blocking calcium influx with calcium channel blockers impaired keratinocyte electrotaxis (Fang et al. 1998). It has been proposed that preferential calcium entry to one side of the cell initiates electrotaxis in that direction (Mycielska and Djamgoz 2004). Other mechanisms are also likely to contribute to electrotaxis, because cathodal migration can occur without cathodal receptor accumulation (Finkelstein et al. 2007) or calcium entry (Brown and Loew 1994).

Although electrotaxis has been mostly studied in in vitro or ex vivo settings, there is evidence that applying EFs in vivo can also trigger electrotactic cell migration. In a mouse model in which CD4 and CD8 T cells express green fluorescent protein (GFP) an EF was applied in the peripheral tissue of the ear in living mice on the stage of a confocal microscope, and the migration of T cells in the ear tissue was recorded (Lin et al. 2008). When exposed to an EF, T cells migrated towards the negative electrode implanted in the ear, moving directionally as much as 100 μm in 1 h. Also, the strength of the EF applied in vivo was between 200 and 500 mV/mm, which is within the same range as EF strengths used in in vitro experiments (McCaig et al. 2005).

Although the use of EFs to guide whole cell migration has not yet reached clinical applications, EFs are actively researched in pre-clinical and clinical settings for their ability to promote nerve outgrowth. EFs have been shown to promote cathodal growth of neurites from frog and mammalian neurons in culture, and subsequently were shown to promote axonal regeneration in animal models of spinal cord injury (McCaig et al. 2005). These results led to a phase 1 trial in humans with spinal cord injury, in which a device generating an oscillating EF was implanted in the site of the wound (Shapiro et al. 2005). The aim of the oscillating device was to promote bidirectional regeneration of nerve fibers, and it reversed the polarity of the EF every 15 min, a time calculated to promote nerve growth towards the cathode on one side of the lesion, but not long enough to induce retraction of nerves facing the anode on the other side. This study led to significant clinical improvement in sensory and motor symptoms, although additional improvement over standard treatments has not been achieved (McCaig et al. 2005).

Neural Stem Cell Migration in Electric Fields

One of the first studies of NSC electrotaxis analyzed the migratory behavior of embryonic neural progenitors in applied EFs (Li et al. 2008). The methodology used was to subject embryonic forebrain explants to EFs, and to analyze the spatial distribution of cells migrating out of the explant. The results obtained indicated that cells leaving the explant were stimulated to migrate with high directionality towards the cathode in the presence of EFs, and that inhibition of the N-methyl-D-aspartate (NMDA) receptor, a glutamate ionotropic receptor, impaired their electrotactic behavior. EF exposure increased a physical association between the NMDA receptor and Tiam1, an activator of the Rac subclass of Rho GTPases, and also increased the phosphorylation levels of Pak1, a kinase involved in actin polymerization downstream of Rac. Therefore, components of the actin remodeling machinery are activated by EFs in a population of neural progenitors, which in turn have a strong directional response to EFs. In this study, a high number of cells co-expressed nestin, a marker of NSCs, and doublecortin, a marker of neuroblasts not found in undifferentiated neural progenitors such as radial glial cells (Noctor et al. 2008), suggesting that the cell population in the embryonic forebrain explants used was a mixture of neural progenitors in different stages of differentiation, most of which seemed nevertheless highly responsive to EF exposure.

Two other studies of NSC electrotaxis (Meng et al. 2011; Arocena et al. 2010) have used both primary cultures of murine embryonic neural progenitors grown initially as neurospheres, which is one of the hallmarks of NSCs, and an extensively studied clone of adult neural stem cells derived from rat hippocampus, which has been previously shown to be able to differentiate into neurons, astrocytes and oligodendrocytes (Kuwabara et al. 2004). Both types of NSCs showed marked electrotactic responses when exposed to EFs of 250 and 500 mV/mm (Meng et al. 2011), migrating towards the cathode. When the polarity of the EF was switched in vitro, cells very quickly reversed course and began migrating in the opposite direction, suggesting that EFs can precisely steer NSC trajectories. Importantly, both electrotaxis and electrotactic reversal were recapitulated in an ex vivo model by transplanting embryonic NSCs into organotypic spinal cord slices and exposing them to EFs, which indicates that environments approaching the complexity of the CNS can support electrotactic migration of NSCs.

Both growth factors and PI3-K signalling are important for NSC electrotaxis (Meng et al. 2011). In the absence of growth factors, electrotactic migration was abolished, although a reduced response remained at higher EF strengths for adult NSCs. There were differences in the growth factor requirements of embryonic and adult NSC electrotaxis, with the former depending on both EGF and FGF, and the latter on FGF only. These are also the growth factors used for routine maintenance of each cell type. Treatment with the PI3-K inhibitor LY294002 markedly reduced electrotaxis in both NSC types, and embryonic NSCs derived from mice lacking the p110γ catalytic subunit of PI3K had a much diminished electrotactic response compared to wild type embryonic NSCs.

A detailed analysis of the migration of adult NSCs in the presence or absence of EFs showed that these cells moved by dynamically extending and retracting protrusions in all directions, but that after an EF was applied protrusion extension was remarkably biased towards the cathode, although this bias was attenuated by treatment with LY294002 (Arocena et al. 2010). This suggested that exposure to EFs was inhibiting the extension of protrusions towards the anode, which was further supported by the observation that protrusions extending towards the cathode retract when the polarity of the EF is reversed. A simple model of NSC electrotaxis based on the experimental data was able to reproduce the migration patterns of NSCs, and suggested that PI3-K functions in NSC electrotaxis mainly, but not only, by controlling the orientation of protrusions.

The propensity of NSCs to migrate in electric fields may represent a potential strategy for clinical targeting towards sites of brain injury, but it is not yet clear for most diseases how this could effectively be achieved in vivo. Nor is it fully apparent under which conditions NSCs could produce clinically significant amelioration of symptoms once they have reached the sites of brain lesion, especially when newly differentiated neurons would have to project and make multiple synaptic connections to restore CNS function. However, sites of brain lesion, which may be associated with perturbation of ion flow (e.g., from damaged cells) and vascularisation, hence producing their own endogenous electric fields, may act as in vivo guidance cues. The response of NSCs to endogenous electric cues can be manipulated genetically as described above and it may be possible therefore to bias their migration clinically. Furthermore, diseases such as multiple sclerosis, that require therapeutic NSCs only to differentiate into oligodendrocytes, offer a more immediate prospect of cell-based therapy based on successful targeting of NSCs to the site of disease.

The main conclusion from these studies is that NSCs are highly responsive to EFs, both in vitro and ex vivo. It is conceivable therefore that devices capable of delivering EFs in vivo, such as the one designed for spinal cord injury treatment, could be used to guide NSC migration, initially in animal models of brain injury, and eventually in clinical settings. NSCs exploit combinations of several directional cues to sustain their remarkable tropism to damaged areas in the CNS. EFs could become a valuable addition to the array of chemotactic, adhesive and topographic signals that NSCs recognize, focussing their migration towards specific targets.