Electrical Stimulation Promotes Stem Cell Neural Differentiation in Tissue Engineering

Nerve injuries and neurodegenerative disorders remain serious challenges, owing to the poor treatment outcomes of in situ neural stem cell regeneration. The most promising treatment for such injuries and disorders is stem cell-based therapies, but there remain obstacles in controlling the differentiation of stem cells into fully functional neuronal cells. Various biochemical and physical approaches have been explored to improve stem cell-based neural tissue engineering, among which electrical stimulation has been validated as a promising one both in vitro and in vivo. Here, we summarize the most basic waveforms of electrical stimulation and the conductive materials used for the fabrication of electroactive substrates or scaffolds in neural tissue engineering. Various intensities and patterns of electrical current result in different biological effects, such as enhancing the proliferation, migration, and differentiation of stem cells into neural cells. Moreover, conductive materials can be used in delivering electrical stimulation to manipulate the migration and differentiation of stem cells and the outgrowth of neurites on two- and three-dimensional scaffolds. Finally, we also discuss the possible mechanisms in enhancing stem cell neural differentiation using electrical stimulation. We believe that stem cell-based therapies using biocompatible conductive scaffolds under electrical stimulation and biochemical induction are promising for neural regeneration.


Introduction
Nerve diseases, including axon loss, nerve injury, and degenerative nerve disease, are a severe economic burden to society. Current medical and surgical strategies and physiotherapy are common treatments for nerve diseases. These strategies alleviate pain after nerve injury, maintain the continuity of nerves, and delay disease progression but are difficult to perform, time-consuming, expensive, and do not always result in sufficient functional recovery and nerve regeneration. Stem cells, including neural stem cells (NSCs) and other exogenous multipotent stem cells, have the ability to differentiate into neural lineages. Accumulating evidence has indicated that stem cell therapy is a promising option in regenerating damaged neurons, assisting functional restoration through the differentiation of stem cells into neurons and glial cells, secreting cytokines and growth factors, acti-vating endogenous repair through immunomodulation, and inhibiting cell apoptosis and fibrosis. In addition, numerous clinical trials have been initiated to evaluate the safety and efficacy of stem cell therapy in patients with various nerve diseases.
A prerequisite in applying stem cells to nerve tissue engineering is controlling the differentiation of stem cells into neural cells with precision and efficacy. Many biophysical strategies, particularly electrical stimulation (ES), have been made to improve the efficiency of stem cell neural differentiation. ES has been demonstrated capable of enhancing the proliferation and differentiation of stem cells, inducing guided cell migration, and promoting the growth and elongation of neurites [1][2][3][4]. In addition, low-frequency ES has also been proven effective clinically in regenerating nerves, hence leading to regeneration and functional recovery [5]; however, the effects of ES on stem cell neural differentiation in different studies slightly vary, owing to the fact that ES frequency, duration, voltage, and the conductive and electroactive material applied varied according to the type of stem cells and loading systems. Thus, the optimal setting for the ES of different stem cells for nerve tissue engineering is difficult to specify. In this review, we summarize various methods in delivering ES to achieve stem cell neural differentiation and maturation both in vitro and in vivo. We also analyse the potential mechanisms of ES in stem cell differentiation. Furthermore, we discuss here our perspectives on the future of the clinical application of ES on stem cells for the treatment of nerve diseases.

Electrical Stimulation Enhances Stem Cell Neural Differentiation
Stem cells can self-renew and differentiate into multiple cell types. In recent decades, many different stem cell types including NSCs, mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), and embryonic stem cells (ESCs) have been investigated in vitro and in vivo to assess the therapeutic potential of stem cell therapies [6][7][8][9][10].
Depending on the origin of stem cells, they exhibit different levels of potency. NSCs located in the specific regions of developing and adult human brain are tissue-specific stem cells and can terminally differentiate into all neural lineages, including neurons, astrocytes, and oligodendrocytes [11]. The application of NSCs is considered a promising therapeutic strategy for treating of central nervous system diseases, including Parkinson's disease, Alzheimer's disease, and spinal cord repair [12][13][14]. Preclinical researches NSCs derived from fetal tissues, ESCs, and iPSCs showed enhanced recovery after stroke [15][16][17] and comparable neurological disorders [18,19]. Due to the similarity of iPSCs and human ESCs (hESCs), similar approaches for the induction of their neural differentiation can be used. Hu et al. compared the neural differentiation capacity between iPSCs and hESCs. They found that iPSCs have the same gene expression pattern and period required to differentiate into functional neurons as ESCs but with increased variability and reduced efficiency [20]. Clinical studies in which ESCs and iPSCs were used for the treatment of nerve diseases are listed in Table 1. At present, most clinical research aims to generate iPSCs from patients with nerve disease to establish disease models, and only a few aim to differentiate iPSCs into neurons and glia for cell transplantation. MSCs are the most commonly used stem cells and can be derived from tissues, such as the bone marrow, adipose tissues, and umbilical cord. Some animal studies have shown that transplanted MSCs can migrate to injured sites of the brain, differentiate into neuron-like cells expressing microtubule association protein-2 (MAP2) and glial fibrillary acidic protein (GFAP), and improve neurological function after stroke and spinal cord injury [21,22]. The differentiation capacity of MSCs from different sources was reportedly not the same. Umbilical cord, bone marrow, and adipose tissue-derived MSCs have been used in clinical research for a number of nerve diseases such as spinal cord injury, amyotrophic lateral sclerosis, and stroke ( Figure 1) [23].
There are complex and varied regulatory networks involved in the neural differentiation of stem cells under different conditions. Certainly, the use of growth factors and small molecules remains the predominant method for stem cell differentiation; however, the use of nonbiochemical methods to assist stem cell differentiation has attracted the attention of many researchers. As neurons are electrically active cells, exogenous ES can provide artificial stimulation that transmits electrical charge directly to the cells. The potential positive effect of exogenous ES on nerve regeneration following injury has been extensively studied. It has been shown that ES can improve neural cell proliferation [24] and the function of neurons and Schwann cells when subjected to a voltage gradient during neural development and postinjury [25]. Exogenous ES has been reported to enhance stem cell neuronal migration [26], differentiation [27], neurite outgrowth [28], and intracellular Ca 2+ dynamics in vitro [29]. Regarding in vivo applications, due to the lack of effective clinical treatments for nerve injuries and neurodegenerative diseases, ES generated from an external power source or from electroactive materials has been explored as a complement and applied in stem cell therapy and tissue engineering since many years ago. Numerous studies on ES therapy have been conducted in animal models and humans and promising results have been reported [30][31][32]. Exogenous ES in animal models not only guides the migration of stem cells and stem cellderived neural cells [33][34][35], but also significantly contributes to stem cell neuron differentiation [36]. In clinical applications, ES therapy as a nonsurgical therapeutic modality is widely adopted by physical therapists and physicians. A variety of ES models have been developed and applied, based on the power sources, including direct current (DC) electric fields, alternating current (AC) electric fields, and pulsed current electric fields. A better understanding of the fundamental principles underlying the ES regulated stem cell neural differentiation would provide clues for developing new strategies for stem cell therapy and devices for nerve tissue engineering.

Effect of Direct Current on Stem Cell Neural
Differentiation. DC indicates that the magnitude and direction of the electric charge is consistent, and it can be produced by batteries, fuel cells, and generators with commutators. Different types of stem cells or their differentiated neuron-like cells respond differently to ES ( Table 2). Min et al. reported that a small DC can guide the migration of human iPSCs (hiPSCs) and hESCs with different electrotaxis depending on distinct signalling pathways. They reported that DC stimulation less than 30 mV/mm guided the migration of hiPSCs to the anode in both twodimensional (2D) and three-dimensional (3D) culture conditions and that the migration rate was voltage-dependent [37], whereas 16 mV/mm DC ES guided the migration of NSCs derived from hESCs to the cathode [35]. In addition, the effect of ES on neural differentiation regulation is cell type specific. The sensitivity of MSCs to the changes in electric field strength was reportedly higher than that of NSCs [38]. More studies are necessary to optimize the parameters of 2 Stem Cells International DC for each stem cell type because of the cell type-specific sensitivity to ES. DC stimulation can also guide NSC migration and enhance NSC differentiation and neural maturation. In a DC electric field of 11.5 V/cm, NSCs tended to specifically differentiate into neurons rather than astrocytes or oligodendrocytes [39]. Kobelt et al. [40] reported that a short duration of ES (10 min/day of DC stimulation at 0.53 or 1.83 V/m) for 2 days enhances neurite outgrowth and βIII-tubulin and neuronal nuclei (NeuN) expression levels and increases the intracellular Ca 2+ during stimulation. The effect of short time ES on stem cell neural differentiation was also confirmed in human MSCs (hMSCs). Greeshma et al. [41] used polyaniline (PANI) to establish conductivity in polymeric substrates and provided a short time DC electric filed stimulation (100 mV/cm, 10 min every day for 10 days). Intermittent ES reportedly improves neural-like differentiation of hMSCs with elongated filopodia and increased expression of nestin and βIII-tubulin [41]. Long-time ES can also enhance stem neural differentiation and maturation. Dong et al. treat NSCs with ES for 3 days at 150 mV/mm, resulting in increased achaete-scute homolog (Ascl1) expression that was further proven to regulate phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway in NSCs [42]. In addition, hMSCs also showed increased levels of SOX2, nestin, βIIItubulin expression, and Ca 2+ oscillation after nine days of continuous exposure to 8 mV/mm ES for 20 h/day [43]. Taken together, these studies confirm the ES can induce cell orientation and migration, and enhance the differentiation of stem cells into neural cell lineages. Since the time duration of ES and the amplitude of DC varied among studies, it is difficult to directly compare the DC-mediated effects on stem cell differentiation reported in them. Besides, none of these studies investigated the effect of ES on neural gene expression profiles throughout the whole process. ES may have variable impacts at different differentiation stages, which warrants further investigation.
Exogenous DC stimulation has also been reported to exert a positive effect on nerve function recovery in vivo. Yamada et al. demonstrated the potential of ESC to 3 Stem Cells International differentiate into mature neurons after injection into the injured spinal cords of adult mice [36]. ES could further improve the function recovery, and 7 days of ES (10 Hz, 0.5-1.0 V), which was performed for 4 h/day, may improve the function of the injured spinal cord in rats [44]. Some data showed that DC stimulation can improve motor function after a stroke [45,46]; in particular, the improvement is greater in chronic stroke patients [47]. The improvements showed a positive relationship with current and charge density when transcranial DC stimulation (tDCS) was applied [48]. Up to 4 mA of tDCS was considered safe and tolerable for stroke patients [49]. In addition, bilateral cerebellar tDCS was also reported to improve balance in patients with Parkinson's disease [50].

Effect of Alternating Current on Stem Cell Neural
Differentiation. AC is the flow of charge that changes direction periodically, and its magnitude reverses along with the current. In vitro AC systems use capacitively coupled or inductively coupled designs. The applications of AC stimulation in neural differentiation are summarized in Table 3. In contrast to DC, AC may not have effect on NSC migration, alignment with ES [51]. This may be due to the bidirectional electric field provided by AC. However, Matos et al. found that AC stimulation can improve the viability and neural differentiation of NSCs. The best frequency for mouse NSC viability was 1 Hz, and frequency lower than 1 Hz can increase the ratio of neurons to astrocytes [52]. Furthermore, using a frequency higher than 1-50 Hz, 0.001 kV/cm, AC ES delayed neural differentiation of progenitor cells into astrocytes [53]. According to these studies, a wide range of the fre-quencies of ES can control the differentiation of stem cells into specific sublineages, which depend on the cell types and culture conditions. Apart from in vitro AC stimulation, in vivo AC devices were also designed. Repetitive transorbital AC stimulation was used to treat mice with optic nerve injury. After treatment, many large neurons survived with moderate dendritic shrinkage [54].

Effect of Pulsed Current on Stem Cell Neural
Differentiation. Pulsed current can be pulsed DC or AC, monophasic or biphasic. Monophasic pulsed current is unidirectional whereas biphasic pulsed current refers to two pulses of current in different directions within one pulse duration or bidirectional. Biphasic current is the most versatile waveform for ES owing to the improved duration, amplitude, and frequency of a pulse. It has been indicated that the parameters of electrical stimulation, including frequency, electrical strength, and duration, should be optimized to improve the effect of ES in regulating stem cell neural differentiation.
Pulsed current has shown remarkable effects on stem cell proliferation, neural differentiation, and axonal outgrowth ( Table 4). Similar to effect of DC on stem cell viability, pulsed ES can improve NSC survival and prevent growth factorinduced cell apoptosis [55]. In addition, the effect of pulsed ES on stem cell proliferation is cell type specific. Petrella et al. [38] compared the effects of picosecond pulsed electric field on NSCs and MSCs. Pulsed ES has no influence on MSC proliferation but improves NSC proliferation and astrocytespecific differentiation by upregulating GFAP after 24 h under 40 kV/cm. Chang KA et al. used [56] used indium tin oxide (ITO) glasses to generate a biphasic electrical stimulator chip. They found that biphasic ES (200 μs pulse duration, 100 Hz) increased not only NSC proliferation but also cell differentiation into NeuN, MAP2, and βIII -tubulin positive neurons. Tandon et al. used a microarray with ITO electrodes to generate monophasic square-wave pulses (5 V, 1 ms duration per 100 ms) and the pulsed ES facilitated mouse retinal progenitor cell differentiation into mature neurons, thereby increasing βIII-tubulin expression and Ca 2 influxes [57].
Pulsed ES also exerts an effect on the differentiation of stem cells into subtypes of neural cells other than neurons. Du et al. reported that 20 Hz of 100 μs pulsed ES enhanced human neural crest stem cell differentiation into Schwann cells and promoted nerve regeneration after cell transplantation [58]. Chang et al. reported that pulsed DC electric fields induce cortical NSCs to simultaneously differentiate into neurons, astrocytes, and oligodendrocytes [59]. In contrast, when NSCs growing on poly (L-lactic-co-glycolic acid) (PLGA)/graphene oxide (GO) conductive composite membranes were stimulated with 500 Hz pulsed current for 1 h every day for 3 days, the NSCs showed differentiation tendency towards neurons comparing to astrocytes [60]. Guo

Effect of Electrical Stimulation through Conductive Material on Neural Differentiation
Restoring nerve function is a great challenge in nerve tissue regeneration. Numerous biomaterials and nanocomponents fulfil the need for achieving the functional differentiation of transplanted stem cells in tissue engineering by mimicking the properties of the microenvironment. Here, we summarize and discuss the electroconductive materials used in nerve tissue regeneration (  [63]. Their data indicated that nanotopography synergistically upregulated the expression of neural markers (Tuj1, NeuN, MAP2) and improved the electrophysiological properties and functional maturation of neurons differentiated from human NSCs.
With the rapid development of biomaterials, conductive polymer materials, including polypyrrole (PPy) [64,65], PANI [66], graphene [67], and carbon nanotubes [68][69][70], have been explored as substrates with acceptable biocompatibility with neural cells. The conductive polymers can locally deliver electrical stimulus to stem cells and even be conjugated with peptides to enhance stem cell proliferation and differentiation. Chuan et al. reported that NSCs planted on a conductive PLGA/GO composite membrane, showed  5 Stem Cells International increased proliferation, neuronal differentiation, and neurite elongation [60]. Peptide-coated PPy neural probes implanted in guinea pig brain promoted the neuron attachment [71]. Ostrakhovitch et al. found that poly(3,4-ethylenedioxythiophene) (PEDOT) : polyethylene glycol (PEG), ITO, and fluorine doped tin oxide (FTO) glass slides can facilitate the neural differentiation of mouse NSE and P19 pluripotent embryonal carcinoma cells and greatly increase the expression of βIII-tubulin [72]. However, Stewart et al. showed that ES in PPy-containing dopant dodecylbenzenesulfonate (DBS) can predominantly induce the differentiation of NSCs into neurons and less likely into glial cells [65]. It remains unclear whether ES can manipulate the differentiation of stem cells into specific subtypes of neurons, including glutamatergic or dopaminergic neurons.
3D printing is an emerging manufacturing technology with great potential in tissue engineering as it provides a powerful fabrication method for generating accurate and complex patterns and architectures with biochemicals and cells. Particularly, 3D printed platforms are being used for neural regeneration [85][86][87]. Hydrogels, biodegradable polymers, and novel biomaterials have been used in 3D printing. Stem Cells International  [77,88,89]. For example, an aqueous dispersion mixture of cellulose nanofibrils (CNF) and single-walled carbon nanotubes (CNT) was used as conductive ink to print guidelines for culturing neural cells (SH-SY5Y) [90]. An amine functionalized MWCNT and polyethylene glycol dipropionate (PEGDA) polymer composite complex was fabricated into a tunable porous neural scaffold that could promote neural stem cell proliferation and neuronal differentiation via a stereolithography 3D printer [77]. Petrella et al. used a 3D printer anchored with a picosecond pulse electric field electrode to print MSCs and NSCs [38]. Their data indicated that 40 kV/cm at 1800 pulses can promote astrocyte specific differentiation but not alter differentiation of MSCs. Aggas et al. also printed 3D hybrid soft conductive hydrogel to support PC12 (a rat pheochromocytoma cell line) attachment [91]. In addition, stem cells and neurites have been shown to grow and extend in the direction of aligned fibers, respectively [92,93]. Differentiated neural cells have been reported to present higher expression levels of neuronal differentiation markers and better properties than random fibers [94][95][96]. In summary, 3D printed conductive nanomaterials offer great advantages for stem cell neural differentiation owing to better morphological control, in addition to biochemical cues.

Potential Mechanism of Electrical Stimulation on Neural Differentiation
In addition to neurotrophic factors, physical stimulation such as ES can also promote neural differentiation. ES can promote stem cell proliferation [24], migration [2], and neuronal differentiation. It regulates the cell differentiation via a complex mechanism, including changes in the extracellular matrix, cell surface receptor activation, microfilament reorganization, Ca 2+ dynamics, and many intracellular signaling pathways. Here, we summarize the potential underlying mechanisms (Figure 3).   Stem Cells International expression is positively related to the strength of the electric field, regulated by ES; this triggers the activation of the PI3K/Akt pathway [42]. In contrast, Rajnicek et al. found that neuronal growth cones migrating toward the cathode were regulated by cell division cycle 42 (Cdc42), Rac, and Rho and not by the PI3K and MAPK/ERK signaling pathways, which were found in the electric field guidance of nonneuronal cells [105]. Electric field-guided directional migration in iPSCs and neurons depends on Rho-kinase signaling [37]. Feng et al. found that a small DC ES (16 mV/mm) was effective in guiding the migration of human ESC-derived NSCs toward the cathode and that this guidance was not exerted through the Rho/Rho-associated protein kinase or C-X-C chemokine receptor type 4 signaling pathway [35]. Wang et al. have found that the brain-derived neurotrophic factor PI3K/Akt signaling pathway activated by BES can protect against growth factor-deprived NSC apoptosis [55]. Moreover, in an in vivo test on rats, ES increased the expression and phosphorylation of ERK1/2 (pERK1/2), and pERK1/2 upregulated the expression of the antiapoptotic protein B-cell lymphoma-2, which finally promoted neuronal cell survival. Furthermore, ES upregulated the expression of p38, which inhibited RhoA-induced neurite outgrowth and neuronal differentiation. These two pathways can lead to the neuronal regeneration and recovery of the electrophysiological function of an injured spinal cord [44]. Chang et al. demonstrated that the combination of nerve growth factor and ES promote neurite outgrowth by increasing the activity of protein kinase C and pERK1/2 [106].
Ca 2+ is an important signaling ion involved in various biological activities. Studies have shown that Ca 2+ influx is important for stem cell fate determination. ES can enhance neural growth toward neurotrophic growth factors by increasing cytoplasmic Ca 2+ and cyclic adenosine monophosphate (cAMP) [107]. Masahisa et al. found that Ca 2+ con-tribute to ES and enhance the neuronal differentiation of ESCs [36]. As a whole, the fundamental mechanism of ESpromoting stem cell neural differentiation is quite complicated, and further research is imperative to completely understand and improve the efficiency of neural regeneration. Coupled with newly developed tools, such as singlecell sequencing and gene editing, these technologies may help identify the ES-induced genes that are crucial for regulating stem cell neural differentiation.

Conclusions
The use of stem cell-derived neural cells is emerging as an effective therapeutic strategy. Stem cells have been used for transplantation to treat nerve diseases with proven safety and efficacy. For example, MSCs have been proven to be safe and effective in treating multiple sclerosis and ischemic stroke [108,109]. Many factors are associated with the efficacy of stem cell therapy and regenerative medicine in nerve diseases. The most important of them is finding effective methods to induce neural differentiation.
Stem cell differentiation is a complicated process that is regulated by various external and internal factors. ES is likely involved in neurogenesis. Compared to the use of chemically or biologically induced differentiation, ES has the advantage of precisely controlling the stimulation through on/off switching and the selective stimulation region as the cells exposed to ES can be easily selected according to the placement of needle electrodes or conductive materials. In addition, ES can be accurately manipulated in a time-controlled manner through an external power supply. However, there are some limitations of ES in the regulation of the neural differentiation of stem cells. Various types of currents, such as DC, AC, and pulsed current, are used in ES; thus, the effects of ES on stem cell differentiation are diverse, depending on the cell types and ES conditions. As such, directly comparing  Figure 3: Potential mechanism of electrical stimulation on neural differentiation. 9 Stem Cells International studies that use different experimental parameters in many aspects is not possible. Importantly, the timing of ES is an essential factor which can strongly influence stem cell differentiation. Nevertheless, current platforms preclude highthroughput screening to simultaneously study the complicated parameters. In addition, although ES is generally effective, it is not as potent as growth factors. This problem can be solved by combining electrical and biochemical stimulation which can potentially promote the differentiation of stem cells in a more robust and controlled manner. Many studies have proved that combined therapy is a strong rational approach for tissue engineering and nerve disease treatment [110][111][112][113]. With the development of materials, micropatterned conductive materials can not only provide ES but also guide cell and neurites orientation through topographies. Some conductive materials, especially nanomaterials, can generate complexed 3D structures to further facilitate scaffold-based cellular transplants. Biomaterial 3D scaffold is one of the most promising approaches for in vivo applications, as it not only can provide a biophysical microenvironment but is also easily compatible with various stimulation cues.
At present, except for orthodox treatment, ES has been considered as a useful noninvasive, interventional method in the clinic. Regardless of the types of waveform of the ES, their effects on neural disease in animal models and human patients have been demonstrated. However, there are many factors that can impact the efficiency of ES-based therapy, such as the source of stem cells, parameters of electric, onset timing and duration of ES, and the stimulation interface materials. A fundamental understanding of the most crucial driving mechanism underlying neural differentiation upon ES will greatly improve the experimental reproducibility and clinical translation. We believe that the combination of new conductive materials and stem cells will contribute to the application of stem cell-based therapy for nerve diseases treatment.

Data Availability
All data included in this study appear in the submitted article.

Conflicts of Interest
The authors declare no competing interests regarding the publication of this paper.