Abstract
Traumatic spinal cord injury (SCI), typically resulting from direct mechanical damage to the spine, often leads to disruption of neural signaling and axonal conduction, severely impairing nervous system function. In rodent models of SCI, neural stem cell (NSC) transplantation has demonstrated significant potential in restoring motor function and enhancing neural repair. Additionally, extremely low-frequency electromagnetic fields (ELF-EMFs) have demonstrated efficacy in promoting nerve regeneration and activating spinal circuits. However, studies exploring how ELF-EMFs influence NSC activation remain limited. In this study, using spinal cord-derived NSCs from adult mice, we report that ELF-EMFs enhance cell proliferation and self-renewal by upregulating Sox2 expression. Furthermore, we addressed the underlying mechanisms and found that ELF-EMFs activate T-type calcium channels and enhance calcium currents. The resulting increase in intercellular calcium concentration upregulates the expression of NeuroG1 and NeuroD1, promoting neuronal differentiation of NSCs and enhancing neurite outgrowth. Our findings provide new insights into the ELF-EMF-mediated activation of NSCs and highlight their potential for integration into combination therapies and SCI repair.
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Introduction
Spinal cord injury (SCI) is a devastating neurological disorder that affects approximately 1.3 million people worldwide, with around 180,000 new cases occurring annually1. SCIs typically result from direct mechanical trauma to the spine, which often triggers secondary immunological responses, leading to neurodegeneration and cytotoxicity2,3. These events disrupt axonal transmission in the nervous system, thereby further compromising neurological function4. Therefore, the key to SCI repair lies in nerve regeneration and neural circuit reconstruction5,6. However, clinical treatments using neurotrophic factors have limited effects on promoting nerve cell regeneration, while neural stem cell (NSCs) transplantation is an effective method6,7. Recently, breakthroughs in NSCs research have provided a strong theoretical basis for SCI repair8,9.
NSCs are multipotent, self-renewing progenitor cells capable of differentiating into specific neurons or glial cells to help restore disrupted neuronal networks10. The delicate balance between NSC proliferation and differentiation is tightly regulated by various factors. For example, Sox2 plays a critical role in maintaining the progenitor state of NSCs11, while the basic Helix-Loop-Helix (bHLH) transcription factor family predominantly governs the differentiation of NSCs12. Previous studies have shown that transplanted NSCs can aid functional recovery in rodent SCI models13,14. Although NSC transplantation has been demonstrated to be clinically safe, the extent of functional recovery and the underlying mechanisms remain insufficiently documented.
Adult endogenous stem cells (AESCs), located in adult neural tissues, have shown potential for regeneration following SCI in mouse and rat models15. Additionally, Weiss et al. discovered that epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) induce the proliferation of spinal neural precursor cells (NPCs)16, confirming the existence of spinal cord-derived NSCs in spinal cord tissue. Although previous studies have revealed that AESCs primarily proliferate into new glial cells in the spinal cord following acute injury15,17,18, the potential for AESCs, especially spinal cord-derived NSCs, to differentiate into neurons, as well as the underlying molecular mechanisms governing this process, remains unclear.
Extremely low-frequency electromagnetic fields (ELF-EMFs), primarily generated by various household appliances, are alternating electromagnetic fields with frequencies below 300 Hz19. During the past few decades, considerable evidence has shown that non-thermal exposure to ELF-EMFs can induce biological changes both in vivo and in vitro20,21, including gene expression22, tissue repair23, and cell proliferation24, all of which are potentially beneficial for treating neurological disorders. Despite accumulating experimental evidence suggesting significant biological effects, the underlying mechanisms remain poorly understood. Previous studies have highlighted the impact of ELF-EMFs at the cellular level25, particularly on the physiological properties of cell membrane and channels26,27,28. Consequently, neurons are likely to be among the most sensitive cells, as exposure to ELF-EMFs may either excite or suppress neuronal activity through interactions with voltage-gated channels27. Calcium influx through voltage-gated calcium channels (VGCCs) has been shown to trigger signaling pathways that regulate gene expression involved in cell proliferation, apoptosis, and neuronal differentiation29. However, the expression and functional role of VGCCs in spinal cord-derived NSCs during ELF-EMF exposure remain poorly understood.
In this study, we provided direct evidence that ELF-EMFs enhance proliferation and self-renewal of spinal cord-derived NSCs by upregulating Sox2. Additionally, ELF-EMFs promote cell differentiation into neurons by activating T-type calcium channels and thereby upregulating pre-neuronal genes, such as NeuroG1 and NeuroD1, which are part of the bHLH transcription factor family. Our findings offer new insights into ELF-EMF-activated functions of NSCs and highlight the potential of combinatorial treatments for clinical applications.
Materials and methods
Mice maintenance
C57BL/6 mice were purchased from Shanghai Sippe-Bk Lab Animal Co., Ltd (Shanghai, China) and housed in a Specific Pathogen Free (SPF) animal facility at Fudan University, operating under Biosafety Level 1 (BSL-1) conditions. Mice were housed in groups of five with a 12-h light/dark cycle, ambient temperature of 22 ± 2 °C, and humidity levels maintained at 40–50%. Food and water were provided ad libitum. All animal experiments in this study were conducted in accordance with the guidelines of the Animal Care and Use Committee of Fudan University and ARRIVE guidelines30, with protocols approved by the Institutional Animal Care and Use Committee of Shanghai, China. Mice used in these studies were euthanized by intraperitoneal injection of pentobarbital (100 mg/kg, 100 mg/ml in normal Saline) using a 27 G needle, confirmed by absence of breathing and heartbeat.
Isolation and culture of spinal cord-derived NSCs
NSCs were isolated from the thoracolumbar spinal cords (T1-L5) of 8-week-old adult C57BL/6 mice using a rigorously optimized protocol31. Following dorsal skin incision and exposure of the vertebral column, the spine was transected caudally and longitudinally incised along the bilateral margins to expose the spinal cord, which was then gently extracted using forceps. To remove erythrocyte contamination, the harvested tissue was treated with red blood cell lysis buffer (Invitrogen, USA). The spinal cords were minced and enzymatically dissociated in 0.25% trypsin-EDTA (Gibco, USA) supplemented with 20 U/ml DNase I (Thermo Scientific, USA) at 37 °C for 10 min. Digestion was terminated with neurobasal medium (Thermo Scientific, USA) containing 10% fetal bovine serum (FBS, Gibco, USA), and the resulting suspension was filtered through a 40-µm cell strainer (Corning, USA) to remove debris. Cells were centrifuged, resuspended, and cultured in proliferation medium. Cells obtained before the first passage (P0) were defined as primary spinal cord-derived NSCs.
NSCs were cultured in proliferation medium, comprising of a 1:1 (v/v) mixture of Dulbecco’s modified Eagle’s medium (DMEM) and F12 medium (Gibco, USA), supplemented with B27 (Gibco, USA), N2 (Gibco, USA), bFGF (20 ng/ml; PeproTech, USA), and EGF (10 ng/ml; PeproTech, USA). The cultures were maintained under floating conditions in 25 cm2 flask with 5 ml of proliferation medium31. Half of the medium was replaced every 3 days. After 7–10 days in vitro, neurospheres were harvested by centrifugation, dissociated using Accutase (Gibco, USA), and collected by centrifugation at 1,000 rpm for 5 min. Cells were resuspended in proliferation medium, and passages two to four (P2-P4) were used for subsequent experiments.
Prior to differentiation, passaged neurospheres were cultured for 1–2 days to allow formation of compact spheres (100–150 μm diameter). To induce differentiation, the NSCs were cultured on poly-D-lysine (PDL)-coated 35 mm dishes in differentiation medium, where bFGF and EGF were replaced with 1% FBS compared to proliferation medium.
ELF-EMF exposure
The system used for exposing cells to electromagnetic fields was as previously described32. Briefly, spinal cord-derived NSCs were exposed to a 50 Hz magnetic field generated by a pair of Helmholtz coils positioned in opposition. The coils were powered by an AC power generator, producing a sinusoidal input voltage, with magnetic flux densities adjustable between 0.1 and 1.0 mT (Fig. 1A). Throughout the exposure period, the duration of ELF-EMF exposure, as well as air and culture medium temperatures, were continuously monitored. Control groups were kept under the same environmental conditions but without ELF-EMF exposure. Based on previous studies33, three different intensities (0.2, 0.5, and 1 mT) were selected, and cells were exposed for 1 h per day for 3 consecutive days (Fig. 1B). Spinal cord-derived NSCs were cultured overnight before being exposed to the ELF-EMFs.
Experimental design and characterization of the spinal cord-derived NSCs. A Schematic of the ELF-EMF system, consisting of a pair of Helmholtz coils powered by an AC generator. B Process diagram for the experiment. Spinal cord-derived NSCs cultured in proliferation medium were exposed to ELF-EMFs for 3 days and then processed for immunocytochemical and molecular analyses, as well as NSCs maintenance assays. Following differentiation induction with proliferation medium containing 1% FBS, NSCs were exposed to ELF-EMFs for 3 days and subsequently processed for immunocytochemical and molecular analyses. C Cultured spinal cord-derived NSCs at generation 0 (P0, left; n = 4). Neurospheres formed by spinal cord-derived NSCs at different passages (P1, middle left; P2, middle right; P5, right; n = 4). Scale bar, 100 μm. D Representative confocal images showing the colocalization of Sox2 (red), Nestin (green), and DAPI (blue) in neurospheres derived from spinal NSCs (n = 5). Scale bar, 25 μm. Insets display magnified view of the boxed region. E Immunostaining for MAP2 (green), GFAP (red), and DAPI (blue) in spinal cord-derived NSCs cultured in differentiation medium for 3 days (n = 4). Scale bar, 100 μm. Insets display magnified view of the boxed region.
Stimulation was performed in 3D neurosphere conditions to assess the effects of ELF-EMFs on undifferentiated NSCs34. Following ELF-EMF exposure, the neurospheres were dissociated into single cells and transferred to 2D adherent conditions for downstream analysis. In the neuronal differentiation assay, NSCs were plated in 2D adherent culture prior to ELF-EMF stimulation to ensure controlled differentiation and reproducible analysis.
Cell proliferation assay
Spinal cord-derived NSCs were cultured in 25 cm2 flasks and exposed to ELF-EMFs at varying intensities for 3 consecutive days. Twenty-four hours after the final exposure, cells were collected, dissociated, and 1.0 × 105 cells were seeded on PDL-coated glass coverslips in 24-well plates. After 4 h of attachment, cells were stained for Ki67 using immunofluorescence.
Neurosphere assays
The neurosphere assays include the neurosphere forming assay (primary NP formation) and the self-renewal assay (secondary NP formation), as previously described35,36. For the primary NP formation assay, single NSCs (1,000 cells in 200 µl proliferation medium) were seeded into a 96-well plate and exposed to ELF-EMFs for 3 consecutive days. Following an additional 4 days of culture, neurospheres were identified and quantified in accordance with strict morphological criteria: phase-bright and spherical structures (100–500 μm in diameter) with surface micro-spikes37. For the secondary NP formation assay, spinal cord-derived NSCs were cultured in proliferation medium and exposed to ELF-EMFs for 3 consecutive days. The newly formed neurospheres were then collected, dissociated into single cells, and seeded into 96-well plates (1,000 cells in 200 µl proliferation medium) without further ELF-EMF exposure. After 7 days of culture, secondary neurospheres were counted using the same stringent morphological criteria. All counts were performed using an inverted microscope (CKX53, Olympus, Japan) at 10× magnification, with data expressed as the mean number of neurospheres per 1,000 seeded cells from ≥ 3 biological replicates.
Immunofluorescent histochemistry
Cells cultured on PDL-coated coverlips were washed twice with PBS and fixed with 4% paraformaldehyde for 15 min. The cells were then permeabilized with 0.5% Triton X-100 in PBS for 30 min at room temperature and blocked with 10% goat serum for 30 min. Coverslips were incubated with primary antibodies (see Table 1) at 4℃ for 24 h, followed by fluorescently conjugated secondary antibodies (Alexa Fluor 488-labelled goat-anti-chicken, Abcam, USA; Alexa Fluor 488-labelled goat anti-mouse, Beyotime Biotechnology, China; Cy3-labeled goat anti-rabbit, Beyotime Biotechnology, China) at room temperature for 1 h. DAPI (Invitrogen, USA) was used to stain the fixed cell nucleus. Cells were counted in 4 different fields of each coverslip using a confocal fluorescence microscope (LSM 700, ZEISS, Germany). The morphometry and the length of neurites were semi-automatically characterized by NeuronJ (ImageJ plugin, ver 1.4.3) according to previous studies38,39.
Western blot analysis
Cells cultured on PDL-coated coverlips were lysed using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, China) supplemented with a protease inhibitor cocktail (Selleck, USA). Protein concentrations were determined via bicinchoninic acid (BCA) assay (Beyotime Biotechnology, China). Protein samples were resolved on 10% SDS-PAGE and transferred to PVDF membranes (Millipore, Germany) using transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) for 90 min at 200 mA. Membranes were blocked with TBS-T (20 mM Tris-HCl adjusted to pH 7.4, 500 mM NaCl, and 0.1% Tween 20) containing 5% non-fat milk at room temperature for 1 h, then probed with specific primary antibodies (see Table 1) overnight at 4 °C. β-actin was used as an internal reference. Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Proteintech, 1 : 5000 in TBS-T with 5% non-fat milk) at room temperature for 1 h. After a final wash with TBS-T, bands were detected by enhanced chemiluminescence (ECL) reagent (Beyotime Biotechnology, China) using an e-Blot Tough Imager (e-Blot, China) following the manufacturer’s instructions. For quantitative analysis, the density of the target bands was quantified using ImageJ software (1.51j8, NIH, USA).
Quantitative real-time PCR
Total RNA was extracted from cells using TRIzol reagent (Takara, Japan). RNA concentrations were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher, USA), with an OD260/OD280 ratio of approximately 2.0. cDNAs were synthesized from 0.5 µg of total RNA using the Transcriptor First Strand cDNA Synthesis Kit (Takara, Japan). Target mRNA levels were quantified via quantitative real-time PCR (qRT-PCR) using SYBR Green Supermix (Bio-Rad, USA) and normalized to GAPDH expression. The specific primers used for qRT-PCR are listed in Table 2.
Whole-cell patch clamp recordings
Whole-cell patch clamp recordings were conducted to examine Ca²⁺ currents, as described previously39. The extracellular solution contained (in mM): 144 tetraethylammonium (TEA)-MeSO3, 10 HEPES, and 1.8 CaCl2, with the pH adjusted to 7.4 using TEA-OH and osmolarity adjusted to 300–310 mOsm with glucose. Recording pipettes (resistance: 4–6 MΩ) were filled with intracellular solution (pH 7.3, adjusted with CsOH) containing (in mM): 138 Cs-MeSO3, 5 CsCl, 0.5 EGTA, 10 HEPES, 1 MgCl2, and 2 mg/ml Mg-ATP. To block T-type calcium channels, cells were exposed to 2 µM TTA-P2 for at least 5 min prior to recording40. Drug application was performed using a gravity-driven perfusion system (VC-6, Warner Instruments) with a flow rate of 2 ml/min, allowing complete solution exchange within 30 s. Following the recording of TTA-P2 effects, a 10-minute washout with TTA-P2-free extracellular solution was conducted to assess reversibility.
Cells were clamped at −70 mV with series resistance compensated by 80%. Recordings were excluded if series resistance exceeded 15 MΩ. Leak currents were subtracted using an online P/4 protocol. Ca²⁺ currents were recorded by depolarizing membrane potentials from −70 to +40 mV in 10 mV increments for 100 ms. Data were obtained using an Axopatch 1550B amplifier (Molecular Device, USA), filtered at 2 kHz, and sampled at 10 kHz.
Data analysis
All data are presented as mean ± SEM (standard error of the mean) from at least 3 independent experiments performed in duplicate, unless otherwise specified. For Gaussian-distributed data, statistical significance was determined using one-way ANOVA with Dunnett’s or Tukey’s post-hoc test. All statistical analyses were performed using GraphPad Prism (version 10.1.2, GraphPad Software, USA) or Igor Pro (version 9.0.5, WaveMetrics, USA). A p-value < 0.05 was considered statistically significant.
Results
Identification of the spinal cord-derived neural stem cells
NPCs were derived from the spinal cords of 8-week-old C57BL/6 mice, following previously published protocols31. After generating a single-cell suspension, the cells were resuspended in proliferation medium for 10 days, leading to the formation of neurospheres (Fig. 1C, left). These neurospheres exhibited self-renewal capacity, as demonstrated by their ability to undergo further subculturing (Fig. 1C). To confirm the identity of the neurospheres, we employed the NSC markers Sox2 and Nestin. Immunostaining showed that the neurospheres were positive for Nestin and Sox2, with the two markers colocalized (Fig. 1D), confirming the presence of NSCs.
In the context of SCI repair, current research and therapeutic strategies primarily emphasize neuronal regeneration and modulation of astrocytic scarring41. To examine the lineage-specific differentiation potential of NSCs toward neurons and astrocytes, dissociated neurospheres were cultured in differentiation medium supplemented with 1% FBS for 3 days, followed by immunostaining for MAP2 and GFAP. MAP2 is specifically localized to dendrites and is critical for microtubule stabilization and neurite extension in post-mitotic neurons42. Its expression has been widely validated across various in vitro neuronal differentiation models and serves as a robust indicator of neuronal identity. GFAP is the principal intermediate filament protein in astrocytes, serving as an astrocyte-specific marker43. After differentiation for 3 days, we observed MAP2⁺ and GFAP⁺ cells, which were biologically exclusive and not co-expressed in the same cell (Fig. 1E). The observed GFAP⁺ and MAP2⁺ cells represent early lineage commitment rather than terminal maturation43,44, indicative of initial fate specification at this early differentiation stage. These results collectively demonstrate that spinal cord-derived NSCs possess the essential characteristics of self-renewal and differentiation into both neurons and astrocytes, which are fundamental properties of NSCs.
ELF-EMFs promote proliferation and self-renewal of spinal cord-derived neural stem cells
To investigate the effects of ELF-EMFs on the proliferation of spinal cord-derived NSCs, cells were exposed to ELF-EMFs at varying intensities (0.2, 0.5, and 1 mT) for 1 h per day over 3 consecutive days (Fig. 1B). The control group was maintained under identical conditions without ELF-EMF exposure (0 mT). Following treatment, the expression of the proliferation marker Ki67 in NSCs was assessed via immunofluorescence staining. A significant increase in the proportion of Ki67+ cells was observed in the ELF-EMF-treated groups compared to the controls (0 mT: 100.0 ± 1.8%; 0.2 mT: 137.8 ± 7.6%, p = 0.048; 0.5 mT: 148.7 ± 13.0%, p = 0.012; 1 mT: 201.7 ± 12.6%, p < 0.001; n = 4; one-way ANOVA with Dunnett’s post-hoc test; Fig. 2A, B), indicating that ELF-EMF exposure promotes the proliferation of spinal cord-derived NSCs.
ELF-EMFs enhance proliferation and self-renewal of spinal cord-derived NSCs. A Representative confocal images showing the proliferation of the spinal cord-derived NSCs stained with Ki67 (green) after exposure to ELF-EMFs at different intensities. Scale bar, 100 μm. Insets display magnified view of the boxed region. B Quantification of Ki67+ cell proportion (n = 4). Values are normalized to the 0 mT control group. C, D Representative images showing self-renewal through primary and secondary neurosphere formation assays after exposure to ELF-EMFs at different intensities. Scale bar, 100 μm. E, F Quantification of the primary (n = 4) and secondary (n = 4) neurosphere formation rates. Values are normalized to the 0 mT control group. G Quantification of Sox2 mRNA levels in spinal cord-derived NSCs after exposure to ELF-EMFs at different intensities (n = 5). Values are normalized to the 0 mT control group.
To investigate whether ELF-EMFs influence the self-renewal of spinal cord-derived NSCs, we performed a neurosphere formation assay. NSCs were digested into single cells and exposed to ELF-EMFs at different intensities (0.2, 0.5, and 1 mT) for 1 h per day over 3 consecutive days. Following exposure, cells were cultured for an additional 4 days to assess the primary neurosphere formation rate (Fig. 1B). The results showed a significant increase in neurosphere formation after ELF-EMF exposure (0 mT: 100.0 ± 6.2%; 0.2 mT: 176.2 ± 8.1%, p = 0.047; 0.5 mT: 212.2 ± 18.1%, p = 0.005; 1 mT: 271.8 ± 33.7% p < 0.001; n = 4; one-way ANOVA with Dunnett’s post-hoc test; Fig. 2C, E), indicating that ELF-EMFs facilitate neurosphere formation in spinal cord-derived NSCs. To assess the maintenance of NSC self-renewal after ELF-EMF exposure, we conducted secondary neurosphere formation analysis. After 3 days of ELF-EMF treatment, newly formed neurospheres were digested into single cells and subcultured for another 7 days to evaluate secondary neurosphere formation (Fig. 1B). The secondary neurosphere formation rate was significantly higher than the control group at 0.5 and 1 mT (0 mT: 100.0 ± 5.2%; 0.2 mT: 131.8 ± 12.9%, p = 0.225; 0.5 mT: 191.6 ± 12.5%, p < 0.001; 1 mT: 215.7 ± 16.8%, p < 0.001; n = 4; one-way ANOVA with Dunnett’s post-hoc test; Fig. 2D, F).
To explore the underlying cellular mechanisms, we extracted RNA from spinal cord-derived NSCs after ELF-EMF exposure and examined the mRNA levels of Sox2 using qRT-PCR. Sox2 is a well-established master regulator of NSC proliferation and self-renewal45. The mRNA levels of Sox2 were significantly upregulated following ELF-EMF exposure (0 mT: 100.0 ± 3.3%; 0.2 mT: 167.5 ± 12.6%, p = 0.048; 0.5 mT: 189.3 ± 23.9%, p = 0.009; 1 mT: 264.6 ± 24.4%, p < 0.001; n = 5; one-way ANOVA with Dunnett’s post-hoc test; Fig. 2G). The upregulation of Sox2 combined with functional assays, such as increased Ki67⁺ cells and enhanced neurosphere formation, suggest that ELF-EMFs enhance and sustain the self-renewal capacity of spinal cord-derived NSCs.
ELF-EMFs facilitate the differentiation of spinal cord-derived neural stem cells into neurons
To facilitate the process of SCI repair, transplanted NSCs must differentiate into neurons or glial cells, as well as form new synaptic connections and neural circuits. Therefore, we investigated the effects of ELF-EMFs on regulating the differentiation ability of spinal cord-derived NSCs into neurons and glial cells. First, we investigated how ELF-EMFs affect neuronal differentiation.
NSCs were induced to differentiate in medium containing 1% FBS and exposed to ELF-EMFs at varying intensities (0.2, 0.5, and 1 mT) for 1 h per day over 3 consecutive days (Fig. 1B). After 3 days, RNA was extracted, and the expression levels of NeuN, a neuron-specific gene, were quantified using qRT-PCR. The results showed that NeuN mRNA levels increased in a dose-dependent manner following ELF-EMF exposure (0 mT, 100.0 ± 6.4%; 0.2 mT: 168.5 ± 19.1%; 0.5 mT: 299.3 ± 22.7%; 1 mT: 321.8 ± 21.0%; 0 mT vs. 0.2 mT, p = 0.078; 0 mT vs. 0.5 mT, p < 0.001; 0 mT vs. 1 mT, p < 0.001; 0.2 mT vs. 0.5 mT, p < 0.001; 0.2 mT vs. 1 mT, p < 0.001; 0.5 mT vs. 1 mT, p = 0.824; n = 5; one-way ANOVA with Tukey’s post-hoc test; Fig. 3C), suggesting that ELF-EMFs facilitate NSCs differentiation into neurons. To further validate this, cells were exposed to ELF-EMFs for 3 days and then immunostained for NeuN (Fig. 3A). The results confirmed that ELF-EMFs facilitate NSCs differentiation into neurons at 0.2 and 0.5 mT (0 mT: 100.0 ± 3.2%; 0.2 mT: 157.1 ± 14.3%; 0.5 mT: 182.0 ± 10.9%; 0 mT vs. 0.2 mT, p = 0.027; 0 mT vs. 0.5 mT, p = 0.002; n = 4; one-way ANOVA with Tukey’s post-hoc test; Fig. 3B). The facilitative effect was slightly reduced at 1 mT (147.2 ± 16.0%; n = 4; 0 mT vs. 1 mT, p = 0.073; Fig. 3B), but was not statistically significant compared to 0.2 and 0.5 mT (0.2 mT vs. 1 mT, p = 0.937; 0.5 mT vs. 1 mT, p = 0.231). Consistent results were obtained from western blot analysis. After 3 days of ELF-EMF exposure, NeuN protein expression was elevated at 0.2 and 0.5 mT (0 mT: 100.0 ± 8.1%; 0.2 mT: 141.2 ± 5.4%; 0.5 mT: 150.9 ± 10.7%; 0 mT vs. 0.2 mT, p = 0.012; 0 mT vs. 0.5 mT, p = 0.002; n = 5; one-way ANOVA with Tukey’s post-hoc test; Fig. 3D, E), while the increase was less pronounced at 1 mT (133.5 ± 7.3%; n = 5; 0 mT vs. 1 mT, p = 0.044; Fig. 3D, E) and not statistically significant compared to 0.2 and 0.5 mT (0.2 mT vs. 1 mT, p = 0.908; 0.5 mT vs. 1 mT, p = 0.454), aligning with the immunostaining data.
ELF-EMFs facilitate differentiation of spinal cord-derived NSCs into neurons. A Representative confocal images showing immunostaining for NeuN after exposure to ELF-EMFs at different intensities. Scale bar, 100 μm. Insets display magnified view of the boxed region. B Quantification of NeuN+ cells after exposure to ELF-EMFs at different intensities (n = 4). Values are normalized to the 0 mT control group. C Quantification of NeuN mRNA levels after exposure to ELF-EMFs at different intensities (n = 5). Values are normalized to the 0 mT control group. D Protein levels of NeuN in spinal cord-derived NSCs after exposure to ELF-EMFs at different intensities, with β-actin used as a loading control. Original blots are presented in Supplementary Fig. 1A. E Quantification of NeuN protein levels after exposure to ELF-EMFs at different intensities (n = 5). Values are normalized to the 0 mT control group. F Representative confocal images stained with Tuj1, showing neurites growth after exposure to ELF-EMFs at different intensities. Scale bar, 50 μm. G Quantification of the number of neurites per cell after exposure to ELF-EMFs at different intensities (n = 5). H Statistics for neurite length per cell after exposure to ELF-EMFs at different intensities (n = 5).
We further investigated the neuronal morphology following ELF-EMF exposure by immunostaining for Tuj1 (neuron-specific class III β-tubulin) to measure neurite number and length. After ELF-EMF exposure, both the number and length of neurites per cell significantly increased at 0.5 mT (number: 2.96 ± 0.13, p = 0.009, n = 5; length: 276.2 ± 16.9 μm, p = 0.009, n = 5; one-way ANOVA with Dunnett’s post-hoc test; Fig, 3F-H). Increases at 0.2 and 1 mT were not statistically significant (number: 0 mT, 2.21 ± 0.14; 0.2 mT, 2.56 ± 0.14, p = 0.295; 1 mT, 2.52 ± 0.20, p = 0.380; n = 5; length: 0 mT, 165.6 ± 15.3 μm; 0.2 mT, 198.9 ± 30.1 μm, p = 0.614; 1 mT, 210.7 ± 25.8 μm, p = 0.390; n = 5; one-way ANOVA with Dunnett’s post-hoc test; Fig. 3F-H). These findings suggest that ELF-EMFs facilitate neurite outgrowth at 0.5 mT, potentially enhancing synaptic connections and neuronal network development.
ELF-EMFs activate pro-neuronal genes and T-type calcium channels
Previous studies have identified key transcription factors of the basic Helix-Loop-Helix (bHLH) family, including Neurogenin1/2 (NeuroG1/2) and Neurogenic Differentiation 1/2 (NeuroD1/2), as critical regulators of neurogenesis46,47. To explore the mechanisms underlying ELF-EMF-induced NSCs differentiation, we assessed the mRNA levels of NeuroD1, NeuroD2, NeuroG1, and NeuroG2 using qRT-PCR. The results showed that ELF-EMF exposure significantly increased the mRNA levels of NeuroD1 and NeuroG1 (Fig. 4A; Table 3), while the mRNA expression of NeuroD2 and NeuroG2 remained unaffected (Fig. 4B; Table 3). Western blot analysis further confirmed increased protein levels of NeuroD1 and NeuroG1 after 3 days of ELF-EMF exposure (Fig. 4C, D; Table 3). These results indicate that ELF-EMFs may facilitate NSC differentiation into neurons by upregulating pro-neuronal gene expression such as NeuroD1 and NeuroG1.
ELF-EMFs upregulate the expression of pro-neuronal genes and activate T-type calcium channels. A, B Quantification of NeuroD1 (n = 4), NeuroG1 (n = 4), NeuroG2 (n = 4), and NeuroD2 (n = 4) mRNA levels in spinal cord-derived NSCs after exposure to ELF-EMFs at different intensities. Values are normalized to the 0 mT control group. C Protein levels of NeuroD1 and NeuroG1 in spinal cord-derived NSCs after exposure to ELF-EMFs at different intensities, with β-actin used as a loading control. Original blots are presented in Supplementary Fig. 1B. D Quantification of NeuroD1 (n = 6) and NeuroG1 (n = 6) protein levels in spinal cord-derived NSCs after exposure to ELF-EMFs at different intensities. Values are normalized to the 0 mT control group. E, F Quantification of Cav1.2 (n = 5), Cav1.3 (n = 5), Cav3.1 (n = 5), Cav3.2 (n = 5) and Cav3.3 (n = 5) mRNA levels in spinal cord-derived NSCs after exposure to ELF-EMFs at different intensities. Values are normalized to the 0 mT control group. G Protein levels of Cav3.1, Cav3.2 and Cav3.3 in spinal cord-derived NSCs after exposure to ELF-EMFs at different intensities, with β-actin used as a loading control. Original blots are presented in Supplementary Fig. 1C. H Quantification of Cav3.1 (n = 3), Cav3.2 (n = 3) and Cav3.3 (n = 3) protein levels in spinal cord-derived NSCs after exposure to ELF-EMFs at different intensities. Values are normalized to the 0 mT control group. I Representative calcium current traces recorded at −30 mV and current density-voltage (I-V) curves of NSC-differentiated neurons after exposure to ELF-EMFs at different intensities (0 mT, black, n = 5; 0.5 mT, green, n = 5; 1 mT, red, n = 5). J Representative calcium current traces recorded at −30 mV and current density-voltage (I-V) curves in NSC-differentiated neurons at 1 mT (1 mT, purple, n = 6; 1 mT + TTA-P2, 1 mT with 2 µM TTA-P2 in the bath solution, blue, n = 6; 1 mT [washout], after 10-min washout with TTA-P2-free solution, light blue, n = 6). K Statistics for peak current density of NSC-differentiated neurons.
Voltage-gated calcium channels (VGCCs) are key mediators of intracellular signaling and have been proposed to be influenced by ELF-EMFs48. Previous studies have shown that calcium influx through VGCC, particularly L- and T-type calcium channels, regulates signaling pathways involved in cell proliferation, programmed cell death, and neuronal differentiation in NSCs48,49,50. To investigate the impact of ELF-EMFs on VGCCs in NSCs, we examined the expression of L- and T-type calcium channels using qRT-PCR. After ELF-EMF exposure, mRNA levels of L-type calcium channels (Cav1.2 and Cav1.3) showed no significant changes (Fig. 4E; Table 3). However, T-type calcium channel (Cav3.1, Cav3.2, and Cav3.3) mRNA levels significantly increased after ELF-EMF exposure (Fig. 4F; Table 3). Western blot analyses further confirmed these findings, showing increased T-type calcium channel protein levels (Fig. 4G, H; Table 3).
To assess the functional activity of calcium channels after ELF-EMF exposure, we recorded calcium currents using whole-cell patch clamp recordings and plotted the current density-voltage (I-V) curve. Whole-cell patch clamp recordings were conducted on day 2 after the final ELF-EMF exposure (Fig. 1B). At this stage, cells had been cultured in differentiation medium for 6 days, exhibiting initial neuronal characteristics. Maximal calcium currents were observed at −30 mV (Fig. 4I), indicating that T-type calcium channels are the predominant VGCCs in NSCs51. After 3 days of ELF-EMF exposure, calcium currents increased significantly without a shift in peak voltage (−30 mV: 0 mT, −1.61 ± 0.45 pA/pF; 0.5 mT, −3.09 ± 0.98 pA/pF; 1 mT, −10.03 ± 1.44 pA/pF; 0 mT vs. 0.5 mT, p = 0.588; 0 mT vs. 1 mT, p < 0.001; 0.5 mT vs. 1 mT, p = 0.001; n = 5; one-way ANOVA with Tukey’s post-hoc test; Fig. 4I, K). This increase was substantially inhibited by 2 µM TTA-P2, a specific T-type calcium channel blocker (−30 mV: 1 mT, −10.23 ± 1.45 pA/pF; 1 mT + TTA-P2, −1.45 ± 1.02 pA/pF; 1 mT [washout], −9.44 ± 1.02 pA/pF; 1 mT vs. 1 mT + TTA-P2, p = 0.004; 1 mT vs. 1 mT [washout], p = 0.878; 1 mT + TTA-P2 vs. 1 mT [washout], p = 0.008; n = 6; one-way ANOVA with Tukey’s post-hoc test; Fig. 4J, K), confirming that ELF-EMFs significantly enhance T-type calcium channel activity.
ELF-EMFs do not affect the differentiation of spinal cord-derived neural stem cells into astrocytes
Glial cells, including astrocytes, oligodendrocytes, and microglia, are abundant non-neuronal cells in the nervous system that support neuronal migration and contribute to nervous system repair and regeneration52. In the injured spinal cord, NSCs largely differentiate into astrocytes, while oligodendrocyte differentiation tends to be limited and typically occurs only in regions with preserved axonal architecture53. To determine whether ELF-EMFs influence astrocytic differentiation, we examined the differentiation of spinal cord-derived NSCs into astrocytes. Astrocytic differentiation was induced using the same protocol as neuronal differentiation, without adding astrocyte-specific cytokines or inducers, allowing for direct comparison of astrocytic and neuronal outcomes.
Immunostaining for GFAP revealed no significant differences in astrocytic differentiation across ELF-EMF intensities from 0.2 to 1 mT (0 mT: 100.0 ± 2.9%; 0.2 mT: 103.7 ± 5.0%, p = 0.963; 0.5 mT: 109.2 ± 10.0%, p = 0.666; 1 mT: 107.3 ± 7.2%, p = 0.794; n = 5; one-way ANOVA with Dunnett’s post-hoc test; Fig. 5A, B). These findings were corroborated by qRT-PCR and western blot analysis, which showed no significant changes in mRNA (0 mT: 100.0 ± 6.7%; 0.2 mT: 89.7 ± 11.6%, p = 0.898; 0.5 mT: 92.7 ± 13.4%, p = 0.958; 1 mT: 107.8 ± 17.7%, p = 0.951; n = 5; one-way ANOVA with Dunnett’s post-hoc test; Fig. 5C) or protein levels (0 mT: 100.0 ± 10.9%; 0.2 mT: 95.8 ± 8.6%, p = 0.991; 0.5 mT, 94.3 ± 11.7%, p = 0.980; 1 mT, 110.8 ± 18.8%, p = 0.889; n = 5; one-way ANOVA with Dunnett’s post-hoc test; Fig. 5D, E), suggesting a negligible effect of ELF-EMFs on astrocytic differentiation from NSCs.
ELF-EMFs have no impact on astrocytic differentiation. A Representative confocal images showing GFAP immunostaining after exposure to ELF-EMFs at different intensities. Scale bar, 100 μm. Insets display magnified view of the boxed region. B Quantification of GFAP+ cells after exposure to ELF-EMFs at different intensities (n = 5). Values are normalized to the 0 mT control group. C Quantification of GFAP mRNA levels in spinal cord-derived NSCs after exposure to ELF-EMFs at different intensities (n = 5). Values are normalized to the 0 mT control group. D Protein levels of GFAP in spinal cord-derived NSCs after exposure to ELF-EMFs at different intensities, with β-actin used as a loading control. Original blots are presented in Supplementary Fig. 1D. E Quantification of GFAP protein levels after exposure to ELF-EMFs at different intensities (n = 5). Values are normalized to the 0 mT control group.
Discussion
The proliferation and differentiation of spinal cord-derived NSCs are essential for maintaining cell populations and generating neuronal diversity during spinal cord development, growth, and repair4,6,8,9. The biological effects of electromagnetic fields, particularly ELF-EMFs, have been studied for over 20 years, with substantial evidence supporting their potential influence on various biological systems54, including cancer55, immune cells23, bone cells56, and nerve cells21,57. Previous studies have shown that ELF-EMF exposure can stimulate cell proliferation across multiple cellular models58, including adult brain NSCs48,59. However, the effects of ELF-EMFs on the proliferation and differentiation of spinal cord-derived NSCs remain poorly understood, thereby limiting their potential clinical applications in SCI treatment.
In this study, we investigated the effects of ELF-EMFs on the proliferation, self-renewal, and multidirectional differentiation of spinal cord-derived NSCs using assays for cell proliferation, neurospheres formation, cell differentiation, neurite analysis, and calcium channel activities. Our findings demonstrate that ELF-EMFs enhance NSC proliferation and self-renewal by upregulating Sox2 expression (Fig. 2). Furthermore, ELF-EMFs significantly promoted neuronal differentiation and increased neurite outgrowth by enhancing T-type calcium channel activity and upregulating pro-neuronal genes NeuroD1 and NeuroG1 (Figs. 3 and 4). However, no significant effect was observed on astrocytic differentiation (Fig. 5).
Previous studies have reported varied effects of ELF-EMFs on neuronal differentiation, with some showing no impact on embryonic NSCs under intermittent exposure60, while others demonstrated enhanced neuronal differentiation in postnatal and adult NSCs under different exposure durations34,48. Our results show that consistent ELF-EMF exposure (0.2, 0.5, and 1.0 mT, 1 h per day for 3 days) significantly increased the proportion of neurons differentiated from spinal cord-derived NSCs (Fig. 3), suggesting that both magnetic field intensity and exposure duration may influence neuronal differentiation. Interestingly, we found that ELF-EMFs did not affect the proportion of NSCs differentiating into astrocytes (Fig. 5), while Isakovic et al. reported that ELF-EMFs steer the differentiation of embryonic NSCs towards astrocyte-like phenotypes18. These findings indicate a selective effect of ELF-EMFs on neuronal lineage development. And this specificity warrants further in vivo investigation to determine whether ELF-EMFs promote spinal cord-derived NSC differentiation preferentially towards neurons or glial cells.
Consistent with the protocol outlined in Fig. 1B, NSCs were collected on day 4 for NeuN mRNA assessment via qRT-PCR immediately after the final ELF-EMF exposure, or on day 5 for NeuN protein detection via immunostaining. We observed that the mRNA levels of NeuN increased in a dose-dependent manner with the intensity of ELF-EMFs (Fig. 3C). However, by day 5, the proportion of neurons in the 0.2 and 0.5 mT groups remained significantly elevated compared to control, while the proportion in the 1.0 mT group showed a decrease relative to its mRNA level (Fig. 3A, B). Western blot analysis of NeuN protein on day 5 also showed reduced expression at 1.0 mT (Fig. 3D, E), suggesting that prolonged exposure to higher ELF-EMF intensities (1.0 mT) may cause cellular damage not evident at earlier time points. These findings indicated that while higher ELF-EMFs intensities promote cell differentiation, they may also compromise neuronal growth due to potential cellular damage.
Neurite initiation is critical for neuronal morphogenesis and early neural circuit formation61, involving nerve fiber projection, synapse formation, and neuronal maturation. Previous studies have reported that ELF-EMF exposure promotes neurite growth in PC12 cells and dorsal root ganglia, with positive correlations to ELF-EMF frequency and intensity34,62. In our study, we found that ELF-EMFs significantly increased the number and length of neurites specifically at 0.5 mT, while the promotional effect was diminished at 1.0 mT (Fig. 4F-H). These findings suggest that neurite outgrowth in spinal NSC-derived neurons is sensitive to ELF-EMF intensity, providing a foundation for potential therapeutic applications of ELF-EMFs in SCI repair. Furthermore, our findings indicate that 0.5 mT represents an optimal condition for inducing neuronal differentiation and neurite outgrowth in spinal cord-derived NSCs.
During the development of the nervous system, the family of bHLH transcription factors, including NeuroG1, NeuroG2, NeuroD1 and NeuroD2, serve as pivotal regulators of neurogenesis, determining neuronal orientation, differentiation, and neural protrusion in embryonic NSCs46,47. A previous study has shown that exposure to 1,800 MHz radiofrequency electromagnetic fields (EMFs) impairs neurite development in embryonic NSCs by downregulating pro-neuronal genes such as NeuroG1 and NeuroD163. However, our results indicate significant upregulation of NeuroG1 and NeuroD1 expression following exposure to ELF-EMFs (50 Hz), highlighting the distinct role of bHLH transcription factors activated by ELF-EMFs in promoting neuronal differentiation and neurite outgrowth in spinal cord-derived NSCs (Fig. 4A-D).
Previous studies have reported that the effects of ELF-EMFs on NSC proliferation and differentiation may be associated with increased VGCCs expression and activity34. For example, in mesenchymal stem cells (MSCs), static magnetic fields promote proliferation by regulating T-type calcium channels and activating the MAPK signaling pathway64. In our study, we found that ELF-EMFs upregulate T-type calcium channel mRNA and protein expression across intensities (Fig. 4F-H), but functional activation of T-type current occurred exclusively at 1 mT (Fig. 4I-K). Given this intensity-restricted functional efficacy, further studies are essential to optimize exposure parameters. We propose that the resulting Ca²⁺ influx mediated by T-type calcium channels subsequently activates the transcription and expression of the pro-neuronal genes, such as NeuroG1 and NeuroD1, promoting neuronal differentiation in NSCs. However, the specific downstream signal pathways driving the activation of pro-neuronal genes warrant further investigation.
Among various calcium channel subtypes, T-type calcium channels are highly expressed during early development and have been shown to modulate NSCs proliferation and neuronal differentiation65. T-type calcium channels are low-voltage-activated calcium channels, responsible for regulating extracellular Ca2+ influx at resting and hyperpolarizing membrane potentials (below −60 mV)29. This distinct gating property makes them particularly responsive in non-excitable cells. Zablotskii et al. reported that magnetic fields can influence ion diffusion via the Lorentz force, potentially altering the membrane potential, which may prevent high-voltage-activated L-type calcium channels from being active66. Consequently, both the unique gating property and upregulated expression levels of T-type calcium channels contribute to the predominant effect of ELF-EMFs. It is plausible that the activation and over-expression of T-type calcium channels could initiate depolarization or Ca²⁺ signaling that promotes the recruitment and activation of other channels like L-type calcium channels or downstream effectors, which, in turn, facilitate NSC differentiation into neurons through Ca2+-mediated signal pathways.
Despite the valuable insights, several limitations of our study must be acknowledged. First, the duration of ELF-EMF exposure was relatively short (1 h per day for 3 consecutive days), and the long-term effects remain unknown. Further studies are required to establish optimal exposure parameters, such as frequency, intensity, and duration, to maximize therapeutic benefits while minimizing potential adverse effects. Second, while this study provides in vitro evidence, it remains unclear whether these exposure parameters are suitable or effective for in vivo applications or whether ELF-EMF-stimulated neurons can restore physiological function. Third, while Sox2 is a well-established master regulator of NSC proliferation and self-renewal, incorporating additional markers, such as HES1/5 or Lsh/HELLS, would strengthen the robustness and generalizability of the transcriptional findings67,68.
In summary, our study demonstrates the potential of ELF-EMFs to specifically activate and promote the proliferation and neuronal differentiation of spinal cord-derived NSCs, presenting valuable insights for SCI repair, regenerative medicine, and neuroscience.
Data availability
All data presented in this manuscript are available from the corresponding author upon reasonable request.
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Acknowledgements
This work was supported by the Science and Technology Innovation 2030 - Brain Science and Brain-Inspired Intelligence Project (2021ZD0201301), the National Key Research & Development Program of China (2022YFC3602700, 2022YFC3602702), the National Natural Science Foundation of China (32170688), the Shanghai Municipal Science and Technology Major Project (2018SHZDZX01), the Shanghai Songjiang District Science and Technology Research Project (22SJKJGG5).
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L.X. and W.X. designed the research; W.X.T. performed most of the experiments and drafted the manuscript; D.H., X.F.L., Y.F., Y.X., and J.W.H. helped with the experiments; L.X. reviewed the manuscript and supervised the project.
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Tang, W., He, D., Li, X. et al. Extremely low-frequency electromagnetic fields facilitate proliferation and functional differentiation in spinal neural stem cells. Sci Rep 15, 29291 (2025). https://doi.org/10.1038/s41598-025-14738-x
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DOI: https://doi.org/10.1038/s41598-025-14738-x