Electrically Preconditioned Engineered Neural Tissues Promote Structural and Functional Repair in Spinal Cord Injury Through Niche-Directed Neural Circuit Reconstruction

Electrically Preconditioned Engineered Neural Tissues Promote Structural and Functional Repair in Spinal Cord Injury Through Niche-Directed Neural Circuit Reconstruction | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Electrically Preconditioned Engineered Neural Tissues Promote Structural and Functional Repair in Spinal Cord Injury Through Niche-Directed Neural Circuit Reconstruction Rongrong Liu, Xiyao Yu, Jiaying Zhou, Zhiyong Dong, Jia Zhao, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7862539/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Spinal cord injury (SCI) creates a prohibitive microenvironment that limits the efficacy of neural stem cell (NSC) therapies. We developed electrically preconditioned engineered neural tissues (ENT) to address these limitations through: (1) pre-establishment of functional neural networks in vitro, and (2) enhanced host integration capacity. EGFP-expressing NSCs were differentiated in 3D Matrigel under 150 mV/mm physiological electric fields (EFs) and transplanted into T10 hemisection SCI mice. Outcomes were assessed through: Basso Mouse Scale (BMS) scoring, multiplex immunofluorescence (Nestin/MAP2/GFAP/MBP/Synaptophysin/ChAT), cortical somatosensory/motor evoked potentials (CSEP/CMEP), RNA sequencing and pathway analysis. We conducted a comprehensive evaluations of the histological structure and function of EF-preconditioned ENT and the mice that received ENT transplantation: (1) in vitro maturation of ENT: high neuronal differentiation, dense synaptic networks and myelinated axon; (2) in vivo integration: niche-directed migration (graft-derived cells showed central canal (Nestin+ cells) and grey matter (ChAT+ cells) homing), achieved functional synaptic integration and correlated with motor recovery. Mechanistic analysis revealed EF activation of pro-neuronal pathways and gliogenesis suppression. These results demonstrate that EF-preconditioned ENT enables structural neural network reconstruction, niche-directed homing, functional synaptic integration and significant motor recovery. Biological sciences/Neuroscience Biological sciences/Stem cells Spinal cord injury Neural stem cells Tissue engineering Bioelectric stimulation Neuronal differentiation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1 Introduction Traumatic spinal cord injury (SCI) causes temporary or permanent impairment of spinal cord function, manifesting as motor, sensory, and autonomic nervous system dysfunction( 1 ). The pathophysiology of SCI involves a cascade of events including neuronal loss, disruption of ascending/descending axonal tracts, ischemia and edema, axonal degeneration, demyelination and cystic cavitation at the injury site( 2 – 5 ). It was well documented that the primary challenges in SCI repair are replacement of lost neurons and re-establishment of functional synaptic connections( 6 – 10 ). Neural stem cells (NSCs) represent an ideal cell source for central nervous system (CNS) repair due to their tri-lineage differentiation potential (neurons, astrocytes, and oligodendrocytes). However, the hostile post-SCI microenvironment often compromises transplanted cell survival and integration( 2 , 11 – 13 ). Animal experiments have shown that transplanted NSCs not only have a high rate of apoptosis, but also lack the differentiation of neurons and oligodendrocytes, resulting in most of the transplanted NSCs differentiating into astrocytes, thereby causing excessive growth of astrocyte scars( 14 , 15 ). Recent advances in engineered neural tissue (ENT) have addressed these limitations. ENT is a 3 dimensional (3D) biofabricated neural tissue derived from NSCs that maintains physiological neural architecture, forms pre-established neural networks, resists inhibitory microenvironmental cues and can be customized in shape/volume for lesion-specific repair( 16 – 18 ). From the perspective of clinical treatment, the optimal ENT for SCI transplantation should contain adequate neuronal numbers, organized neurite outgrowth, functional synaptic networks and myelinated fibers( 19 , 20 ). A number of studies, including our previous research, have shown that electrical stimulation (ES) has significant potential in regulating or controlling the following events: ( 1 ) promoting neuronal differentiation of NSCs( 21 ); ( 2 ) guiding the parallel arrangement of neurites in 3D cultures( 22 ); ( 3 ) stimulating branching of neurites; ( 4 ) accelerating synaptogenesis and myelination. Therefore, the synergistic effect of EF with other factors may play a crucial role in constructing an ENT with well-developed neural network( 23 – 26 ). In our previous studies, we utilized EFs to stimulate mNSCs within Matrigel droplets. The EF stimulation provided the appropriate bioactive cues, thereby successfully constructing ENT with an appropriate number of neuronal cells, highly branched neurites, and well-developed neuronal network. These structural features of ENT indicate that it is a relatively mature neural tissue with a certain 3D organizational structure and a stable microenvironment( 27 , 28 ). Building on this foundation, we generated EGFP-labeled ENT using EF stimulation (150 mV/mm), transplanted it into a murine SCI model and systematically evaluated cell survival and migration, differentiation patterns, synaptic integration and functional recovery. Our results indicated that EF stimulation enhances neuronal yield, synaptogenesis and myelination in ENT. This 3D structural ENT provides mechanical support vs cell suspensions and pre-differentiation bypasses hostile host microenvironment. We demonstrated for the first time that EF-preconditioned ENT exhibits niche-directed migration (central canal and grey matter homing), achieves functional synaptic integration and correlates with motor recovery. 2 Methods 2.1 Animal care 66 C57BL/6 mice (Adult, female, 20 ~ 25 g) were used for all animal experiments in this study. All experiments were carried out in accordance with the Institutional Animal Care and Use Committee guidelines at Jilin University. Animals had free access to food and water throughout the study. All surgical procedures were performed under anaesthesia produced by inhalation with Isoflurane. Animal experiments were approved by the Ethics Committee of Experimental Animals in Basic Medical College of Jilin University ((2025) Study No. 411). EGFP transgenic mice were purchased from Cyagen Biotechnology ( EGFP Tg/+ mice, Cyagen Biotechnology Co., LTD, Suzhou, China). 2.2 Materials Key cell culture reagents, including Accutase, HEPES buffer, and the B27 and N2 supplements, were sourced from Gibco (CA, USA). Growth factors, specifically human recombinant bFGF and EGF (both at 20 ng ml − 1 ), were acquired from PeproTech (NJ, USA). The basal medium DMEM/F12, along with penicillin/streptomycin, fetal bovine serum (FBS), and Ca/Mg-free PBS, were all procured from HyClone (SU, USA). Corning (NY, USA) supplied the 75 µm cell strainers and Growth Factor-Reduced Matrigel. Fisher Scientific (NH, USA) provided bovine serum albumin (BSA) and fetal calf serum. A range of reagents for cell coating, staining, and mounting—including Poly-D-lysine, laminin, gelatin, TRITC-conjugated phalloidin, DAPI, and CC/Mount—were obtained from Sigma Aldrich (SL, USA). Additionally, Triton-X was purchased from VWR (PA, USA), and the nuclear stain Hoechst 33342 was supplied by Solarbio (Beijing, China). 2.3 Primary culture of mNSCs and EGFP Tg/+ mNSCs Following a previously established protocol( 27 ), primary embryonic mNSCs were isolated from the 13 to 16-day-old embryos of C57BL/6 mice or EGFP Tg/+ mice, and then expanded in a specific growth medium. In brief, fetal brain tissues underwent mechanical dissociation to form a single-cell suspension, which was subsequently cultured in DMEM/F12 medium. This medium was enhanced with an N2 supplement, human recombinant bFGF (20 ng ml − 1 ) and human recombinant EGF (20 ng ml − 1 ). Cells were seeded at a density of 5,000 cells per cm² to facilitate neurosphere formation. During passaging, neurospheres were digested with Accutase to obtain single cells, which were then replated under the same culture conditions. Phenotypic identification was performed using immunocytochemistry to evaluate the expression of NSC markers, Nestin and Musashi1, as well as differentiation markers, Tuj1 and GFAP. 2.4 Electrical stimulation-induced 3D mENT We adopted the electrotactic chamber and hENT construction protocol from previous studies( 27 , 29 ), with slight adaptations. In brief, neurosphere suspensions were resuspended in ice-cold Matrigel at a 1:5 ratio (neurospheres: Matrigel) and mixed thoroughly under chilled conditions. This mixture was then polymerised by incubating it at 37°C. The resulting matrix was overlaid with BNb medium, which consists of DMEM/F12 supplemented with N2 and B27 additives, in addition to 20 ng ml − 1 recombinant human bFGF. Cultures experienced a direct current electric field (DC EF) of 150 mV mm − 1 for 30 minutes each day over a three-day period. Post-stimulation, the 3D constructs were carefully detached and relocated to new Petri dishes for suspension culture. These constructs remained in an orbital shaker incubator at 85 rpm until transplantation, with the medium renewed every 2–3 days. For structural and phenotypic validation, the mENTs were analyzed via confocal microscopy following immunocytochemical staining, enabling the generation of high-resolution, surface-rendered 3D reconstructions. 2.5 Immunofluorescence assay mNSCs were plated evenly in poly-L-lysine-coated petri dishes. After being treated with 4% paraformaldehyde for 20 minutes, the samples were washed three times with PBS then treated with 0.3% Triton X-100 for 10 minutes. The samples were washed three times with PBS to remove any excess permeating fluid. After being treated with 5% BSA blocking buffer, the primary antibodies (Table S1 ) were added. After overnight incubation at 4°C, the samples were washed three times, and then the secondary antibody (Alexa Fluor 488-, 594- or 555-conjugated anti-mouse, anti-rabbit or anti-chicken IgY, 1:100; Life Technology, OR, USA) was incubated for 60 minutes. For 3D-cultured samples, the immunofluorescence protocol followed a previously established method( 27 ). Key differences from the 2D protocol included an extended fixation step (12 hours in 4% PFA at 4°C) and the use of a specialized blocking solution (PBS with 0.1% Triton X-100, 2% BSA, and 2% gelatin) for 30 minutes. This was followed by permeabilization with 0.5% Triton X-100 in PBS for 30 minutes and subsequent incubation with primary antibodies (Table S1 ) at 4°C overnight. After extensive washing with the blocking solution, samples were incubated with fluorescently tagged secondary antibodies (as listed above, used at 1:200) overnight at 4°C, followed by an extended PBS wash. Finally, all samples (both 2D and 3D) were counterstained with Hoechst33342 for 10 minutes before image acquisition. 2.6 Electron microscopy technique for detecting ultrastructure of mENT Sample preparation for transmission electron microscopy followed a previously established protocol ( 27 ). In brief, the 3D cultures were primarily fixed by overnight immersion at 4°C in a solution of 2% paraformaldehyde and 3% glutaraldehyde in PBS. After thorough rinsing with PBS, the samples were post-fixed in 1% osmium tetroxide for one hour at room temperature. Dehydration was then performed using a graded ethanol series, followed by embedding in pure epoxy resin and polymerization at 60°C. Subsequently, 50 nm ultrathin sections were cut and double-stained with uranyl acetate and lead citrate to enhance contrast. Finally, the sections were visualized under a transmission electron microscope (EP5018/40/Tecnai Spirit Biotwin 120KV, FEI Czech Republic s.r.o, The Netherlands). 2.7 Surgical procedures Mice were randomly assigned to four experimental groups: a sham-operated group (sham surgery, n = 6), a spinal cord injury (SCI) group (normal saline administration, n = 20), an ENT transplantation group (n = 20), and a neural stem cell (NSC) transplantation group (n = 20). Following anesthesia, a laminectomy was performed at the T9-T11 vertebral levels. A spinal cord hemisection injury model was then created by gently excising tissue using microscissors, resulting in a lesion approximately 3 mm in width and 1 mm in depth. Subsequently, the surgical incisions were sutured, and the mice were returned to their home cages. All animals received comprehensive post-operative care, which consisted of daily intraperitoneal injections of 80,000 units of penicillin for 5 consecutive days and manual bladder expression twice daily until reflexive bladder function was restored. All surgical procedures and animal handling protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Jilin University and conducted in strict compliance with its guidelines. 7 days post-SCI, motor function was assessed in all mice using the 21-point Basso Mouse Scale (BMS; see Table S2 for details). Mice exhibiting consistent and severe functional deficits—specifically, complete hindlimb paralysis (absence of movement, Babinski reflex score of 0) and loss of the bladder emptying reflex—were selected for inclusion in the final SCI model cohort. ENT grafts were transplanted to fill the lesion cavities produced by the hemisection. A suspension of NSCs was injected into the injury site using a 5-µl Hamilton syringe (Hamilton Company, Reno, NV, USA). 2.8 Electrophysiological analysis 28 days post-transplantation, 6 mice per group were anesthetized and secured in a stereotaxic apparatus. After exposing the sciatic nerve and cerebral cortex, electrodes from a BL-420E Data Acquisition and Analysis System (Tai-meng Technology, China) were positioned on the exposed sciatic nerve and cerebral cortex. The latency and amplitude of both cortical somatosensory evoked potentials (CSEPs) and cortical motor evoked potentials (CMEPs) were recorded. 2.9 Histology analysis At 7 days, 14 days and 28 days after transplantation, intracardiac perfusion was performed on all animals, using 4% paraformaldehyde (PFA) as the perfusion fluid. The isolated spinal cord tissues were fixed in 4% PFA for 24 hours, treated with a 5% sucrose solution for 1–2 hours, a 15% sucrose solution overnight, and a 30% sucrose solution until tissue pieces settle to the bottom for cryoprotection. Cut into 15-millimeter-thick sagittal sections using a cryostat. Hematoxylin-eosin (HE) staining was performed for general histological examination. The immunostaining procedure followed the previously described( 28 ). Primary antibodies used are listed in Table S1 . Secondary antibodies included Alexa Fluor 488-conjugated goat anti-mouse, anti-rabbit IgG, or anti-chicken IgY, as well as Alexa Fluor 594/555-conjugated goat anti-mouse or anti-rabbit IgG (all used at 1:200 dilution; Life Technologies, Carlsbad, CA, USA). Imaging was performed using a fluorescence microscope (Olympus IX71, Japan) and a confocal microscope (FV3000, Olympus, Japan). Z-stack confocal imaging was acquired using the Olympus FV3000 microscope. ImageJ software (National Institutes of Health, https://imagej.nih.gov/ij/ ) or its plugin (3D viewer) was used to analyze the fluorescence intensity and generate surface rendered 3D images. 2.10 Statistical analyses Statistical analysis was conducted using Origin Parametric software. Data is expressed as mean ± standard deviation. Statistical analyses were performed using one-way ANOVA and Fisher’s Test. Statistical significance was defined as * p < 0.05, ** p < 0.01, and *** p < 0.001. 3 Results 3.1 Construction of Mouse Engineered Neural Tissue (mENT) in Vitro Building on our previous work demonstrating that 150 mV/mm physiological DC EFs induce neuronal differentiation in 3D Matrigel cultures( 27 , 28 ), we generated EGFP-expressing mENT (EGFP-ENT) to track graft integration. EGFP-NSCs (characterized in Figure S1 ) were suspended in Matrigel and exposed to DC EFs (150 mV/mm, 30 min/day for 3 consecutive days), followed by 4 days of differentiation. 3D reconstruction imaging revealed that the EGFP-ENT constructs with consistent diameters (4 ~ 5 mm; Fig. 1 A) and thicknesses (180 ~ 300 µm; Fig. 1 B and E). At 7 days post-differentiation, immunostaining showed predominant neuronal commitment, with 72.49 ± 3.0% of EGFP + cells expressing the early neuronal marker Tuj1, compared to only 8.94 ± 2.5% GFAP + astrocytes (Fig. 1 C). By day 14, neuronal maturation increased further, with 74.8 ± 5.0% of cells expressing the mature marker MAP2, while GFAP + astrocytes declined to 6.21 ± 2.2% (Fig. 1 D). Within the 3D tissue, neurites formed elaborate networks (Fig. 1 F) with abundant synaptophysin + puncta (Fig. 1 G). Transmission electron microscopy (TEM) confirmed ultrastructural hallmarks of functional neural tissue, including vesicle-rich presynaptic terminals (Fig. 1 K), thickened pre- and postsynaptic densities (Fig. 1 I, J), and myelinated axons (Fig. 1 H), indicating advanced neural tissue maturation. 3.2 Superior Structural Stability of 3D ENT in Large-volume lesions The experimental design and timeline are summarized in Fig. 2 A. Sixty-six adult female C57BL/6 mice were randomized into four groups, including a group that received laminectomy only (sham-operated group, n = 6), a SCI group that received normal saline (SCI group, n = 20), a SCI group that received EGFP-ENT transplantation (ENT group, n = 20), and a SCI group that received EGFP-NSC transplantation (NSC group, n = 20). Histological analysis confirmed successful T9-11 hemisecting injuries, characterized by cavitation and inflammatory infiltration (Figure S2). Small-animal imaging at 24 hours post-transplantation verified graft placement, with both EGFP-ENT and EGFP-NSCs filling the lesion cavity (Fig. 2 B and C). By 7 days post-transplantation (dpt), the ENT group maintained dense EGFP + cell populations organized into intact tissue structures (Fig. 2 D), whereas NSC grafts showed minimal EGFP + cell retention (Fig. 2 E), demonstrating the superior structural stability of 3D ENT in large-volume lesions. 3.3 Transplantation of EGFP-ENTs Enhances Locomotor and Electrophysiological Recovery in SCI Mice BMS assessments revealed progressive recovery across all groups (Fig. 3 A). While the SCI group reached a plateau at 4.1 ± 2.0 by 28 days post-transplantation (dpt), the EGFP-ENT group achieved significantly higher scores (7.3 ± 0.8 vs EGFP-NSC: 5.6 ± 2.6; p < 0.01). Gait analysis showed distinct recovery patterns: 1 day post-SCI (dpi): Complete hindlimb paralysis in all injured groups; 5 dpi: EGFP-ENT mice developed ankle movement (SCI: no volitional movement); 7 dpi: EGFP-ENT mice showed consistent foot-ground contact (SCI: only sporadic limb movement); 10 ~ 14 dpi: EGFP-ENT mice achieved weight-bearing (SCI: occasional standing with instability); 21 ~ 28 dpi: EGFP-ENT mice demonstrated coordinated plantar stepping (SCI: no coordinated movement) (Fig. 3 B, Video S1-4). Cortical somatosensory/motor evoked potentials (CSEP/CMEP) measurements showed: 24 h post-SCI: 80% reduction in amplitude (vs sham) and latency increased by 150% (vs sham). 28 dpt: ENT group: Near-normal amplitudes (90% of sham) and latencies (110% of sham); NSC group: Partial recovery (60% amplitude, 130% latency vs sham); SCI group: Minimal improvement (Fig. 3 C-E). These results demonstrate that EGFP-ENT transplantation significantly enhances both structural and functional repair after SCI compared to single-cell suspensions. 3.4 Spatiotemporal Dynamics of EGFP-ENT Integration in Host Spinal Cord We systematically evaluated graft survival, migration, and synaptic integration at 7, 14, and 28 dpt using immunofluorescence for lineage-specific markers (GFAP, MAP2, MBP, ChAT and synaptophysin) and NSC marker Nestin. Migration distances were quantified from the lesion epicenter (n = 3 mice/timepoint). 3.4.1 Short-Term Engraftment (7 dpt) EGFP-ENT grafts showed EGFP-ENT filled into the lesion site (Fig. 4 A), with cells migrating into host tissue. Migrating EGFP + cells exhibited dual differentiation: astrocytic lineage: GFAP + cells at graft-host interface (Fig. 4 B); neuronal lineage: MAP2 + cells in lesion site and white matter (Fig. 4 C). Notably, no EGFP+/MAP2 + cells were detected in gray matter (Fig. 4 D), indicating stage-limited integration. 3.4.2 Intermediate Phase (14 dpt) Migration distance increased to 1.25 ± 0.31 mm, with three distinct populations: central canal homing (EGFP+/MAP2- clusters) (Fig. 5 A-C), white matter penetration (EGFP+/MAP2 + neurons) (Fig. 5 D), gray matter entry (EGFP+/SYP- cells) (Fig. 5 E and F). It is worth noting that at this stage, the cells entering the grey matter have not yet expressed synaptophysin. 3.4.3 Long-Term Integration (28 dpt) Only a few EGFP + cells remained at the graft core (Fig. 6 A), maturated graft-derived EGFP+/MAP2 + neurons exhibited in gray matter (Fig. 6 B) and interacted with host neurons, which can be identified by the presenting of both EGFP+/SYP+ (yellow arrows) and EGFP-/SYP + cells (white arrows) (Fig. 6 C-F). By analyzing the ultrastructure of the spinal cord after injury and transplantation of ENT, we found that the spinal cord tissue at the injury site was structurally disordered, with vacuoles appearing within the cells, and the myelin sheath structure became thinner and incomplete (Fig. 6 H) compared to the normal spinal cord tissue (Fig. 6 G). On the contrary, in the individuals receiving ENT transplantation, the structure of the spinal cord injury center was relatively compact (Fig. 6 I). Long protrusions were seen to be arranged in parallel (Fig. 6 J). At the injury site, intact myelin sheaths (Fig. 6 K) and larger blood vessels invading (Fig. 6 L) could be observed. These results indicate that bioelectric stimulation generates 3D ENT with pre-formed neuronal cells. After transplantation into spinal cord lesions, ENT-derived cells exhibit progressive migration and differentiation into mature neurons, and functional synaptic integration with host circuits, enabling motor recovery. 3.5 Niche-Directed Homing and Lineage Diversification of ENT-Derived Cells At 14 dpt, spatial analysis revealed a striking compartmentalization of EGFP + cell fate determination (Fig. 7 ), with three distinct microanatomical distributions. First, central canal homing guided by the stem cell niche engagement. NSC Retention: migrated EGFP + cells formed periventricular clusters, exhibiting strong Nestin co-expression (Fig. 7 A). Second, reconstruction of the motor circuit dominates the differentiation of spinal gray matter cells. Motor neuron specification: gray matter-infiltrating EGFP + cells expressed choline acetyltransferase (ChAT), displaying characteristic large somata and ventral horn localization (Fig. 7 B), suggesting microenvironment-driven lineage bias. Third, oligodendrogenesis and myelination potential promotes white matter integration. We also detected MBP-positive engrafted cells in the spinal cord white matter (Fig. 7 C), exhibiting mature, multi-lamellar sheaths (Fig. 6 K). These results implicated the unique characteristics of ENT transplantation: ( 1 ) Niche-specific recruitment: central canal homing mirrors endogenous NSC trafficking, implying conserved chemoattractant pathways; ( 2 ) functional diversification: simultaneous ChAT + neuron and MBP + oligodendrocyte generation demonstrates multi-lineage reparative potential. 3.6 Transcriptomic Mechanisms of EF-Induced Neuronal Differentiation 3.6.1 RNA-Seq Profiling of EF-Stimulated 3D ENT To elucidate the molecular basis of EF-enhanced neurogenesis, we performed RNA sequencing on 3D ENT cultures following 7 days of EF stimulation (150 mV/mm, 30 min/day) versus unstimulated controls (n = 3 replicates/group). After stringent quality control (Q30 > 90%, Phred score ≥ 30), reads were aligned to GRCm38/mm10 using STAR (v2.7.10b), achieving 92.4 ± 2.1% mapping efficiency. Differential expression analysis revealed that there are 375 significantly regulated genes (adjusted p < 0.05) out of 12,661 detected transcripts (Fig. 8 A and B). Downregulated Genes (n = 174) are enriched for gliogenic (Gfap ↓2.1-fold) (Fig. 8 C).The upregulated Genes (n = 201) are dominated by neurodevelopmental regulators (Fig. 8 D) like Nrgn (Neurogranin, calcium-calmodulin signaling modulator, ↑8.2-fold, p = 3.2×10⁻⁷) and Camk2a (Synaptic differentiation mediator, ↑5.6-fold, p = 1.8×10⁻⁵). 3.6.2 Functional Annotation and Pathway Enrichment Analysis ClusterProfiler software performed GO functional enrichment analysis (Fig. 9 A) and KEGG pathway enrichment analysis of the differential gene set (Fig. 9 B). For results of GO functional enrichment, a p-value < 0.05 was used as the threshold for significant enrichment. ClusterProfiler identified three key functional clusters (Fig. 9 A) including: ( 1 ) Biological Processes: synapse assembly (FDR = 2.1×10⁻⁷) and neuron projection development (FDR = 4.3×10⁻⁶) ( 2 ) Cellular Components: dendritic spine (FDR = 7.8×10⁻⁵) and presynaptic active zone (FDR = 1.2×10⁻⁴); ( 3 ) Molecular Functions: voltage-gated calcium channel activity (FDR = 3.5×10⁻³) and tubulin binding (FDR = 5.1×10⁻³). KEGG pathway enrichment (Fig. 9 B) mainly included the following pathways (top enriched pathways, q < 0.01): Calcium signaling (8 genes, q = 2.4×10⁻⁴), PI3K-AKT (7 genes, q = 6.1×10⁻⁴), Wnt (5 genes, q = 8.3×10⁻³), Axon guidance (6 genes, q = 9.7×10⁻³). 3.6.3 Protein-Protein Interaction (PPI) Network From the 375 DEGs, PPI analysis (STRING DB) revealed that there are 367 nodes and 331 edges (interaction enrichment p = 1.0×10⁻¹⁶). Among the identified genes, we extracted and presented five hub genes that are closely associated with calcium ion channels and neural differentiation. (Refer to Fig. 10 ): Camk2a (degree = 28), Pik3r1 (PI3K regulatory subunit, degree = 25), Cacna1a (voltage-gated calcium channel, degree = 22), Dvl1 (Wnt signaling, degree = 19) and Ntrk2 (BDNF receptor, degree = 17). Key Mechanistic Insights Based on the above analysis, we propose a potential mechanism of the regulation of neuronal differentiation by EFs. EF stimulation activates a calcium-PI3K/Wnt signaling axis that drives neuronal commitment while suppressing glial fate. Synaptogenic programs are initiated prior to transplantation, priming ENT for functional integration. Hub gene topology suggests EF mimics endogenous neurotrophic signaling (BDNF/NT-3 pathways). Discussion Spinal cord injury initiates a pathological cascade involving inflammatory infiltration, glial scarring, and cavitation, leading to axonal degeneration and functional impairment( 30 , 31 ). While NSC transplantation has shown promise in SCI repair( 2 ), its efficacy is limited in severe cases due to poor cell survival within large lesion cavities and low neuronal differentiation rate( 32 – 34 ) . Our study addresses four critical barriers to repair: ( 1 ) Microenvironment modulation: 3D ENT structure mitigates inhibitory signals; ( 2 ) Cell replacement: pre-differentiated neurons bypass host differentiation blockade; ( 3 ) Circuit integration: pre-formed neurons and oligodendrocytes enable rapid host connectivity; ( 4 ) Niche-directed homing and lineage diversification of ENT-derived cells. In this study, the application of physiological EFs induced the pre-differentiation of NSCs in 3D matrix in vitro, resulting in the construction of mature neural tissues with a high proportion of neurons and a large number of synapses and myelin sheaths, forming a well-developed neural network within the ENT. This mature 3D neural tissue has a self-regulating microenvironment( 35 ). Thus, it can possess a certain ability to resist the inhibitory microenvironment of damaged spinal cord, facilitating its survival and continued growth in the host, and also greatly shortens the time from stem cells to neurons, reduces the risks of abnormal proliferation( 9 , 36 ). Unlike previous NSC differentiation studies under 3D environment reporting 70% neurons, likely due to EF-induced PI3K /AKT/β-catenin activation, which suppresses glial fate( 27 , 38 ). On the other hand, transplantation in the acute phase of injury was used not only to avoid excessive hyperplasia of glial scar and further deterioration of microenvironment after SCI, but also to reduce the damage to mice caused by multiple surgeries( 39 ). Compared to conventional NSC therapy( 40 ), our approach provides some advantages: ( 1 ) Structural protection: providing 3D structural support to reduce mechanical stress; ( 2 ) Pre-differentiation: pre-differentiating NSCs into neurons before transplantation, avoiding hostile microenvironment effects; ( 3 ) Network maturity: accelerating functional integration through pre-differentiated neurons and synaptic connections. The BMS score results showed that within 28 days, the functional recovery speed of mice transplanted with EGFP-ENT was faster than that of the group without EGFP-ENT transplantation, which also indicates that the transplantation of EGFP-ENT does indeed help the functional recovery of SCI mice. Notably, we investigated spatiotemporal integration dynamics of ENT in SCI mice. 7 dpt: Radial dispersion with dual GFAP+/MAP2 + differentiation; 14 dpt: Niche homing; 28 dpt: Functional synapses on ChAT + motor neurons. This homing behavior likely results from chemoattractants (e.g., SDF-1/CXCR4 axis( 41 )) secreted by the canal microenvironment, suggesting ENT-derived NSCs may synergize with host stem cells to enhance repair. The homing of EGFP+/Nestin⁺ cells to the central canal—a niche for endogenous NSCs—suggests their potential to recruit host stem cells for repair( 42 , 43 ). In this study, not only showed NSCs homing behavior, but also mature neurons. At 28 dpt, we detected typical ChAT-positive motoneurons in the gray matter of the spinal cord, with large cell bodies, large round nuclei, well-developed processes, and numerous synaptophysin-positive sites around the cell bodies and processes. ChAT-positive neurons were motor neurons located in the anterior horn of the gray matter of the spinal cord, indicating that the neurons in EGFP-ENT migrate to the interior of the spinal cord, while continuing to differentiate and mature into different neuronal subtypes and migrate to specific locations to establish synaptic connections with different cells. This may be the structural basis for ENT transplantation to promote functional recovery in mice with SCI( 44 ). We further investigated the mechanistic insights of EF-regulated neuronal differentiation. Transcriptomics revealed EF-activated pathways which include Calcium-CAMK2A: drives axonal growth (↑8.2-fold Nrgn); PI3K-AKT: enhances survival (↑5.6-fold Pik3r1); Wnt/β-catenin: suppresses glial fate (Gfap ↓2.1-fold)( 7 ). In conclusion, we have developed a novel cell transplantation therapy that uses electrical stimulation to pre-build 3D neural tissues for the treatment of SCI. The therapeutic effect of this treatment is significantly better than that of NSC treatment. ENT transplantation promoted the recovery of the mice's motor function in the short term and established neural circuits (CSEP/CMEP significantly improved). One limitation of this study is that, compared with traditional spinal cord contusion or resection surgeries, we used an uncommon SCI model, which enabled us to transplant the corresponding volume of ENT and evaluate the histological changes in the spinal cord injury and the transplantation area. Additionally, avoiding the death of graft cells due to immune rejection reactions remains a major challenge after transplantation( 45 ). This study only observed the grafts for a short period (28 days), and the long-term survival of the graft cells requires the use of immunosuppressive drugs to solve this problem, which requires long-term observation in the future( 46 ). Overall, although this study has certain limitations, electrical stimulation of pre-differentiated ENT transplantation will still become a new approach for treating SCI, and it may also provide new ideas and methods for tissue engineering transplantation to treat central nervous system injuries. Conclusion Our ENT platform achieves structural and functional restoration through pre-formed neural networks; it realizes the synergy between the host and the graft through specialized and niche-adaptive homing mechanisms and provides clinically valuable targets (the calcium-PI3K-Wnt axis). The current limitations include temporal mismatch: there is a difference between the recovery time of mice (28 days) and that of humans (several months to several years)( 47 ), and spatial scaling issues: a porcine model is needed to simulate the relevant distances in humans. Future work will focus on large animal validation and the production of ENT in accordance with GMP standards. Declarations The authors declare that they have not use AI-generated work in this manuscript Ethics approval and consent to participate Animal experiments were approved by the Ethics Committee of Experimental Animals in Basic Medical College of Jilin University ((2025) Study No. 411). Competing interests All authors have declared no conflict of interest in this manuscript. Funding This work was supported by grants from Department of Science and Technology of Jilin Province: Key Scientific and Technological Research and Development Projects (No. 20180201026YY), to XTM. Author Contribution XTM and FW designed the study. XTM and XYY wrote the manuscript. RRL made the figures, carried out most experiments and interpreted results. XYY analyzed the transcriptome results. JYZ carried out animal experiments. XTM and FW conceptualized and supervised the experiments. ZYD, JZ and ZCW completed final editing and revision of the manuscript. 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Supplementary Files Supplementarymaterial.zip Graphicalabstract.png Graphical abstract Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 28 Dec, 2025 Reviews received at journal 19 Dec, 2025 Reviews received at journal 06 Dec, 2025 Reviewers agreed at journal 26 Nov, 2025 Reviewers agreed at journal 14 Nov, 2025 Reviewers invited by journal 14 Nov, 2025 Submission checks completed at journal 13 Nov, 2025 First submitted to journal 13 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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14:05:34","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":484229,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7862539/v1/e69673b003af9da266b30ddd.png"},{"id":96740384,"identity":"4df2a8ee-aab1-4bfa-bac2-5e70447354ec","added_by":"auto","created_at":"2025-11-25 14:54:22","extension":"png","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":172568,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7862539/v1/99027cd58cefe5a3bc7c02e2.png"},{"id":96740392,"identity":"9bca396f-af7c-4d16-b740-300415b749b9","added_by":"auto","created_at":"2025-11-25 14:54:22","extension":"png","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":83848,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7862539/v1/85ea8ff1c24e71ffcade5865.png"},{"id":96740395,"identity":"21cf9d5c-4936-46f5-9f22-61602e1e81be","added_by":"auto","created_at":"2025-11-25 14:54:23","extension":"xml","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":129938,"visible":true,"origin":"","legend":"","description":"","filename":"35f76712cc384222a1b683cb25c00edf1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7862539/v1/e7a99a102e40d884b3fe8dab.xml"},{"id":96914149,"identity":"ef15ff88-1177-40ad-b7c8-662128303271","added_by":"auto","created_at":"2025-11-27 14:05:32","extension":"html","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":143123,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7862539/v1/d9269ad9bfe976470e350481.html"},{"id":96740356,"identity":"b6761870-48ab-48a1-a9a9-2346407b345d","added_by":"auto","created_at":"2025-11-25 14:54:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1200930,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of 3D EGFP-ENT. \u003c/strong\u003e(A) Macroscopic view of EGFP-ENT construct. (B) 3D reconstruction showing tissue architecture. (C) Quantification of neuronal (Tuj1+) and glial (GFAP+) differentiation at day 7 (n=5 independent cultures). (D) Maturation profile at day 14 showing MAP2+ neurons and GFAP+ cells. (E) Immunofluorescence (IF) staining of mature neurons (MAP2, red), nuclei (Hoechst, blue), and EGFP-positive cells (green) (scale bar = 200 μm). (F) High-magnification view of interconnected neurites (scale bar = 20 μm). (G) Synaptic puncta (Synapsin, red) among EGFP+ cells (scale bar = 50 μm). (H-K) Transmission electron microscopy (TEM) images showing: (H) Myelinated axons (scale bar = 200 nm), (I) Presynaptic terminals, (J) Postsynaptic densities (scale bar = 200 nm), and (K) Synaptic vesicles (scale bar = 100 nm). Yellow arrows highlight key structures.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7862539/v1/0a63f873c49c15d22a068a3b.png"},{"id":96914513,"identity":"70ba051a-981a-44d2-8374-bed25439b888","added_by":"auto","created_at":"2025-11-27 14:06:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":857986,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe EGFP-ENTs or NSCs were implanted in mice undergoing T9-11 spinal cord hemi-section.\u003c/strong\u003e (A) Experimental timeline showing EGFP-ENT construction, SCI surgery (Day 0), transplantation (Day 7), and analysis endpoints. (B) shows the intact spinal cord without the graft. (C) Both transplant types (EGFP-ENTs and EGFP-NSCs) filled the lesions in the host spinal cord. (D) Lesion site filling by EGFP-ENT and (E) EGFP-NSCs at 7 dpt (scale bars, left = 200 μm, right= 100 μm). Dashed lines indicate lesion boundaries.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7862539/v1/a3fc709cf6eee7bcb9eb0fa7.png"},{"id":96914382,"identity":"fd35d994-d1b3-4cc9-b98e-f71590dff0d6","added_by":"auto","created_at":"2025-11-27 14:05:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":529294,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional recovery after EGFP-ENT transplantation. \u003c/strong\u003e(A) BMS scores over 28 days post-transplantation (dpt). ENT group shows significant improvement vs NSC and SCI groups (*\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; two-way RM ANOVA). (B) Representative gait patterns. (C-E) Electrophysiological recovery: (C) Somatosensory evoked potential (CSEP) and Cortical motor evoked potential (CMEP); (D-E) Latency comparisons at 28 dpi. Data are mean ± SEM (n=6/group). * \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01,*** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7862539/v1/8f523463e8ab071377d2f757.png"},{"id":96740361,"identity":"cd8dfb19-ea2a-42e3-b104-8e4bef690685","added_by":"auto","created_at":"2025-11-25 14:54:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1032696,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eShort-term graft integration at 7 dpt \u003c/strong\u003e(A)\u0026nbsp;EGFP-ENT survival at lesion site (scale bar 25 = μm).\u0026nbsp;(B)\u0026nbsp;Dual-labeled EGFP\u003csup\u003e+\u003c/sup\u003e/GFAP\u003csup\u003e+\u003c/sup\u003e astrocytes (arrow) at interface (scale bar = 50 μm).\u0026nbsp;(C)\u0026nbsp;EGFP\u003csup\u003e+\u003c/sup\u003e/MAP2\u003csup\u003e+\u003c/sup\u003e neurons in white matter (arrow) (scale bar = 50μm).\u0026nbsp;(D)\u0026nbsp;Absence of EGFP\u003csup\u003e+\u003c/sup\u003e/MAP2\u003csup\u003e+\u003c/sup\u003e cells in gray matter (scale bar = 50 μm).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7862539/v1/3c311e2a34448551bd59fab6.png"},{"id":96740362,"identity":"610f236b-f119-41e4-b2bc-57f45deaed04","added_by":"auto","created_at":"2025-11-25 14:54:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1247596,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntermediate-phase migration patterns at 14 dpt \u003c/strong\u003e(A, B)\u0026nbsp;EGFP\u003csup\u003e+\u003c/sup\u003e cells remaining at the lesion site. EGFP\u003csup\u003e+ \u003c/sup\u003ecells infiltrate into\u0026nbsp;(C)\u0026nbsp;central canal (pericentral EGFP\u003csup\u003e+\u003c/sup\u003e/MAP2\u003csup\u003e-\u003c/sup\u003e clusters) and\u0026nbsp;(D)\u0026nbsp;gray matter (EGFP\u003csup\u003e+\u003c/sup\u003e/MAP2\u003csup\u003e+\u003c/sup\u003e clusters).\u0026nbsp;(E, F) EGFP\u003csup\u003e+\u003c/sup\u003e/SYP\u003csup\u003e-\u003c/sup\u003e cells (arrow) entry gray matter. Yellow dashed lines indicate anatomical boundaries.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7862539/v1/2de404d173b05959bbd13453.png"},{"id":96740363,"identity":"9e4120f5-f474-4611-b5b1-3853173cf83c","added_by":"auto","created_at":"2025-11-25 14:54:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1697980,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLong-term synaptic integration at 28 dpt \u003c/strong\u003e(A)\u0026nbsp;Graft dispersion pattern (scale bar = 200 μm).\u0026nbsp;(B)\u0026nbsp;Co-existing EGFP\u003csup\u003e+\u003c/sup\u003e/MAP2\u003csup\u003e+\u003c/sup\u003e (yellow arrows) and host (white arrows) neurons (scale bar = 20 μm).\u0026nbsp;(C-E)\u0026nbsp;Confocal z-stacks showing SYP\u003csup\u003e+\u003c/sup\u003e puncta (yellow arrows) on EGFP\u003csup\u003e+\u003c/sup\u003e neurons (C: scale bar = 50 μm; D-E: scale bar = 20 μm). (G) well organized normal spinal cord tissue (scale bar = 2 μm). (H) structurally disordered tissue in injury center (scale bar = 2 μm). (I) shows the junction where the transplanted ENT meets the host spinal cord tissue (scale bar = 10 μm). The surrounding spinal cord tissue of the transplanted ENT shows long protrusions arranged in parallel (J) (scale bar = 1 μm), intact myelin sheaths (K) (scale bar = 1 μm) and larger blood vessels (L) (scale bar = 10 μm).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7862539/v1/e977b76b786042cf968b5a50.png"},{"id":96740374,"identity":"18881dbd-dbdb-47b0-8065-2e844b45e62a","added_by":"auto","created_at":"2025-11-25 14:54:22","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":666965,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpatially Resolved Fate Mapping of ENT-Derived Cells at 14 and 28 dpt\u003c/strong\u003e. (A)\u0026nbsp;EGFP\u003csup\u003e+\u003c/sup\u003e/Nestin\u003csup\u003e+\u003c/sup\u003e neural stem cells (green/red) clustered within the central canal niche (outline) (scale bar = 50 μm).\u0026nbsp;\u003cem\u003eInset\u003c/em\u003e: showing radial alignment. (B)\u0026nbsp;EGFP\u003csup\u003e+\u003c/sup\u003e/ChAT\u003csup\u003e+\u003c/sup\u003e motor neurons (green/red) in ventral horn gray matter (scale bar = 20 μm). (C)\u0026nbsp;MBP\u003csup\u003e+\u003c/sup\u003e myelinating oligodendrocytes (green/red) in corticospinal tract (scale bar = 20 μm). (D)\u0026nbsp;Rare EGFP\u003csup\u003e+\u003c/sup\u003e/GFAP\u003csup\u003e+\u003c/sup\u003e astrocytes (green/red) (scale bar = 5 μm).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7862539/v1/88812a07a46fbf579ee4a1f8.png"},{"id":96914716,"identity":"fa036027-b0cd-4430-b1ca-294bca8ee08b","added_by":"auto","created_at":"2025-11-27 14:06:16","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":673504,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic Landscape of EF-Stimulated NSC. \u003c/strong\u003e(A) Volcano plot showing 375 significant DEGs (red: upregulated; blue: downregulated; |log2FC|\u0026gt;1, padj\u0026lt;0.05) (EF vs noEF). (B) Heatmap of top 20 upregulated neurogenic genes. (C) Gliogenic gene suppression. (D) The upregulated Genes (n=201) are dominated by neurodevelopmental regulators. Data from n=3 biological replicates.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7862539/v1/b0cdd8668a2bf322d2c31721.png"},{"id":96740368,"identity":"35b3ebf4-2e48-47e2-9aa4-c6b6b78101aa","added_by":"auto","created_at":"2025-11-25 14:54:22","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":769650,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePathway Enrichment Analysis. \u003c/strong\u003e(A) GO terms (BP, CC, MF) with top 10 enrichments per category. Circle size indicates gene count; color shows -log10 (FDR). (B) KEGG pathway network. Edges represent shared genes between pathways.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7862539/v1/6c7ee4f8dfc6eb54e91c29e7.png"},{"id":96914300,"identity":"ab92fad3-0ae0-4e31-94b0-ebc1e4ccfa24","added_by":"auto","created_at":"2025-11-27 14:05:41","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":206253,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePPI Network of Neuronal Differentiation Hub Genes\u003c/strong\u003e. Cytoscape-generated interaction network (confidence score \u0026gt;0.7). Node size reflects degree centrality; colors indicate pathway association: gold (calcium), blue (PI3K-AKT), green (Wnt).\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-7862539/v1/51f736a3e19987e3836246a0.png"},{"id":96922179,"identity":"30b4986b-afe9-4381-91cc-80bf00dfbd71","added_by":"auto","created_at":"2025-11-27 14:18:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10118154,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7862539/v1/4af87d9f-4c54-41fb-b6c5-33deeb4db922.pdf"},{"id":96740396,"identity":"04abb541-44cc-41b8-9a87-a732b9bb2f01","added_by":"auto","created_at":"2025-11-25 14:54:23","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18012394,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.zip","url":"https://assets-eu.researchsquare.com/files/rs-7862539/v1/a3c6c487504dc819397255d5.zip"},{"id":96914566,"identity":"c5c276fa-61d7-4167-b140-b735dfc2fdd7","added_by":"auto","created_at":"2025-11-27 14:06:03","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":438698,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract\u003c/p\u003e","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-7862539/v1/e8387e21c3183c19d7a5ca98.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Electrically Preconditioned Engineered Neural Tissues Promote Structural and Functional Repair in Spinal Cord Injury Through Niche-Directed Neural Circuit Reconstruction","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTraumatic spinal cord injury (SCI) causes temporary or permanent impairment of spinal cord function, manifesting as motor, sensory, and autonomic nervous system dysfunction(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). The pathophysiology of SCI involves a cascade of events including neuronal loss, disruption of ascending/descending axonal tracts, ischemia and edema, axonal degeneration, demyelination and cystic cavitation at the injury site(\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). It was well documented that the primary challenges in SCI repair are replacement of lost neurons and re-establishment of functional synaptic connections(\u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eNeural stem cells (NSCs) represent an ideal cell source for central nervous system (CNS) repair due to their tri-lineage differentiation potential (neurons, astrocytes, and oligodendrocytes). However, the hostile post-SCI microenvironment often compromises transplanted cell survival and integration(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Animal experiments have shown that transplanted NSCs not only have a high rate of apoptosis, but also lack the differentiation of neurons and oligodendrocytes, resulting in most of the transplanted NSCs differentiating into astrocytes, thereby causing excessive growth of astrocyte scars(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRecent advances in engineered neural tissue (ENT) have addressed these limitations. ENT is a 3 dimensional (3D) biofabricated neural tissue derived from NSCs that maintains physiological neural architecture, forms pre-established neural networks, resists inhibitory microenvironmental cues and can be customized in shape/volume for lesion-specific repair(\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). From the perspective of clinical treatment, the optimal ENT for SCI transplantation should contain adequate neuronal numbers, organized neurite outgrowth, functional synaptic networks and myelinated fibers(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eA number of studies, including our previous research, have shown that electrical stimulation (ES) has significant potential in regulating or controlling the following events: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) promoting neuronal differentiation of NSCs(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e); (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) guiding the parallel arrangement of neurites in 3D cultures(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e); (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) stimulating branching of neurites; (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) accelerating synaptogenesis and myelination. Therefore, the synergistic effect of EF with other factors may play a crucial role in constructing an ENT with well-developed neural network(\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn our previous studies, we utilized EFs to stimulate mNSCs within Matrigel droplets. The EF stimulation provided the appropriate bioactive cues, thereby successfully constructing ENT with an appropriate number of neuronal cells, highly branched neurites, and well-developed neuronal network. These structural features of ENT indicate that it is a relatively mature neural tissue with a certain 3D organizational structure and a stable microenvironment(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBuilding on this foundation, we generated EGFP-labeled ENT using EF stimulation (150 mV/mm), transplanted it into a murine SCI model and systematically evaluated cell survival and migration, differentiation patterns, synaptic integration and functional recovery. Our results indicated that EF stimulation enhances neuronal yield, synaptogenesis and myelination in ENT. This 3D structural ENT provides mechanical support vs cell suspensions and pre-differentiation bypasses hostile host microenvironment. We demonstrated for the first time that EF-preconditioned ENT exhibits niche-directed migration (central canal and grey matter homing), achieves functional synaptic integration and correlates with motor recovery.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"2 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Animal care\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e66 C57BL/6 mice (Adult, female, 20\u0026thinsp;~\u0026thinsp;25 g) were used for all animal experiments in this study. All experiments were carried out in accordance with the Institutional Animal Care and Use Committee guidelines at Jilin University. Animals had free access to food and water throughout the study. All surgical procedures were performed under anaesthesia produced by inhalation with Isoflurane. Animal experiments were approved by the Ethics Committee of Experimental Animals in Basic Medical College of Jilin University ((2025) Study No. 411).\u003c/p\u003e\u003cp\u003eEGFP transgenic mice were purchased from Cyagen Biotechnology (\u003cem\u003eEGFP\u003c/em\u003eTg/+ mice, Cyagen Biotechnology Co., LTD, Suzhou, China).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Materials\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eKey cell culture reagents, including Accutase, HEPES buffer, and the B27 and N2 supplements, were sourced from Gibco (CA, USA). Growth factors, specifically human recombinant bFGF and EGF (both at 20 ng ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), were acquired from PeproTech (NJ, USA). The basal medium DMEM/F12, along with penicillin/streptomycin, fetal bovine serum (FBS), and Ca/Mg-free PBS, were all procured from HyClone (SU, USA). Corning (NY, USA) supplied the 75 \u0026micro;m cell strainers and Growth Factor-Reduced Matrigel. Fisher Scientific (NH, USA) provided bovine serum albumin (BSA) and fetal calf serum. A range of reagents for cell coating, staining, and mounting\u0026mdash;including Poly-D-lysine, laminin, gelatin, TRITC-conjugated phalloidin, DAPI, and CC/Mount\u0026mdash;were obtained from Sigma Aldrich (SL, USA). Additionally, Triton-X was purchased from VWR (PA, USA), and the nuclear stain Hoechst 33342 was supplied by Solarbio (Beijing, China).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Primary culture of mNSCs and \u003cem\u003eEGFP\u003c/em\u003e\u003csup\u003eTg/+\u003c/sup\u003emNSCs\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFollowing a previously established protocol(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), primary embryonic mNSCs were isolated from the 13 to 16-day-old embryos of C57BL/6 mice or \u003cem\u003eEGFP\u003c/em\u003eTg/+ mice, and then expanded in a specific growth medium. In brief, fetal brain tissues underwent mechanical dissociation to form a single-cell suspension, which was subsequently cultured in DMEM/F12 medium. This medium was enhanced with an N2 supplement, human recombinant bFGF (20 ng ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and human recombinant EGF (20 ng ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Cells were seeded at a density of 5,000 cells per cm\u0026sup2; to facilitate neurosphere formation. During passaging, neurospheres were digested with Accutase to obtain single cells, which were then replated under the same culture conditions. Phenotypic identification was performed using immunocytochemistry to evaluate the expression of NSC markers, Nestin and Musashi1, as well as differentiation markers, Tuj1 and GFAP.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Electrical stimulation-induced 3D mENT\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eWe adopted the electrotactic chamber and hENT construction protocol from previous studies(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), with slight adaptations. In brief, neurosphere suspensions were resuspended in ice-cold Matrigel at a 1:5 ratio (neurospheres: Matrigel) and mixed thoroughly under chilled conditions. This mixture was then polymerised by incubating it at 37\u0026deg;C. The resulting matrix was overlaid with BNb medium, which consists of DMEM/F12 supplemented with N2 and B27 additives, in addition to 20 ng ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e recombinant human bFGF. Cultures experienced a direct current electric field (DC EF) of 150 mV mm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for 30 minutes each day over a three-day period. Post-stimulation, the 3D constructs were carefully detached and relocated to new Petri dishes for suspension culture. These constructs remained in an orbital shaker incubator at 85 rpm until transplantation, with the medium renewed every 2\u0026ndash;3 days. For structural and phenotypic validation, the mENTs were analyzed via confocal microscopy following immunocytochemical staining, enabling the generation of high-resolution, surface-rendered 3D reconstructions.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Immunofluorescence assay\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003emNSCs were plated evenly in poly-L-lysine-coated petri dishes. After being treated with 4% paraformaldehyde for 20 minutes, the samples were washed three times with PBS then treated with 0.3% Triton X-100 for 10 minutes. The samples were washed three times with PBS to remove any excess permeating fluid. After being treated with 5% BSA blocking buffer, the primary antibodies (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) were added. After overnight incubation at 4\u0026deg;C, the samples were washed three times, and then the secondary antibody (Alexa Fluor 488-, 594- or 555-conjugated anti-mouse, anti-rabbit or anti-chicken IgY, 1:100; Life Technology, OR, USA) was incubated for 60 minutes.\u003c/p\u003e\u003cp\u003eFor 3D-cultured samples, the immunofluorescence protocol followed a previously established method(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Key differences from the 2D protocol included an extended fixation step (12 hours in 4% PFA at 4\u0026deg;C) and the use of a specialized blocking solution (PBS with 0.1% Triton X-100, 2% BSA, and 2% gelatin) for 30 minutes. This was followed by permeabilization with 0.5% Triton X-100 in PBS for 30 minutes and subsequent incubation with primary antibodies (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) at 4\u0026deg;C overnight. After extensive washing with the blocking solution, samples were incubated with fluorescently tagged secondary antibodies (as listed above, used at 1:200) overnight at 4\u0026deg;C, followed by an extended PBS wash. Finally, all samples (both 2D and 3D) were counterstained with Hoechst33342 for 10 minutes before image acquisition.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Electron microscopy technique for detecting ultrastructure of mENT\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eSample preparation for transmission electron microscopy followed a previously established protocol (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). In brief, the 3D cultures were primarily fixed by overnight immersion at 4\u0026deg;C in a solution of 2% paraformaldehyde and 3% glutaraldehyde in PBS. After thorough rinsing with PBS, the samples were post-fixed in 1% osmium tetroxide for one hour at room temperature. Dehydration was then performed using a graded ethanol series, followed by embedding in pure epoxy resin and polymerization at 60\u0026deg;C. Subsequently, 50 nm ultrathin sections were cut and double-stained with uranyl acetate and lead citrate to enhance contrast. Finally, the sections were visualized under a transmission electron microscope (EP5018/40/Tecnai Spirit Biotwin 120KV, FEI Czech Republic s.r.o, The Netherlands).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Surgical procedures\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eMice were randomly assigned to four experimental groups: a sham-operated group (sham surgery, n\u0026thinsp;=\u0026thinsp;6), a spinal cord injury (SCI) group (normal saline administration, n\u0026thinsp;=\u0026thinsp;20), an ENT transplantation group (n\u0026thinsp;=\u0026thinsp;20), and a neural stem cell (NSC) transplantation group (n\u0026thinsp;=\u0026thinsp;20). Following anesthesia, a laminectomy was performed at the T9-T11 vertebral levels. A spinal cord hemisection injury model was then created by gently excising tissue using microscissors, resulting in a lesion approximately 3 mm in width and 1 mm in depth. Subsequently, the surgical incisions were sutured, and the mice were returned to their home cages. All animals received comprehensive post-operative care, which consisted of daily intraperitoneal injections of 80,000 units of penicillin for 5 consecutive days and manual bladder expression twice daily until reflexive bladder function was restored. All surgical procedures and animal handling protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Jilin University and conducted in strict compliance with its guidelines. 7 days post-SCI, motor function was assessed in all mice using the 21-point Basso Mouse Scale (BMS; see Table S2 for details). Mice exhibiting consistent and severe functional deficits\u0026mdash;specifically, complete hindlimb paralysis (absence of movement, Babinski reflex score of 0) and loss of the bladder emptying reflex\u0026mdash;were selected for inclusion in the final SCI model cohort. ENT grafts were transplanted to fill the lesion cavities produced by the hemisection. A suspension of NSCs was injected into the injury site using a 5-\u0026micro;l Hamilton syringe (Hamilton Company, Reno, NV, USA).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Electrophysiological analysis\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e28 days post-transplantation, 6 mice per group were anesthetized and secured in a stereotaxic apparatus. After exposing the sciatic nerve and cerebral cortex, electrodes from a BL-420E Data Acquisition and Analysis System (Tai-meng Technology, China) were positioned on the exposed sciatic nerve and cerebral cortex. The latency and amplitude of both cortical somatosensory evoked potentials (CSEPs) and cortical motor evoked potentials (CMEPs) were recorded.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Histology analysis\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAt 7 days, 14 days and 28 days after transplantation, intracardiac perfusion was performed on all animals, using 4% paraformaldehyde (PFA) as the perfusion fluid. The isolated spinal cord tissues were fixed in 4% PFA for 24 hours, treated with a 5% sucrose solution for 1\u0026ndash;2 hours, a 15% sucrose solution overnight, and a 30% sucrose solution until tissue pieces settle to the bottom for cryoprotection. Cut into 15-millimeter-thick sagittal sections using a cryostat. Hematoxylin-eosin (HE) staining was performed for general histological examination. The immunostaining procedure followed the previously described(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Primary antibodies used are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Secondary antibodies included Alexa Fluor 488-conjugated goat anti-mouse, anti-rabbit IgG, or anti-chicken IgY, as well as Alexa Fluor 594/555-conjugated goat anti-mouse or anti-rabbit IgG (all used at 1:200 dilution; Life Technologies, Carlsbad, CA, USA).\u003c/p\u003e\u003cp\u003eImaging was performed using a fluorescence microscope (Olympus IX71, Japan) and a confocal microscope (FV3000, Olympus, Japan). Z-stack confocal imaging was acquired using the Olympus FV3000 microscope. ImageJ software (National Institutes of Health, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imagej.nih.gov/ij/\u003c/span\u003e\u003cspan address=\"https://imagej.nih.gov/ij/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) or its plugin (3D viewer) was used to analyze the fluorescence intensity and generate surface rendered 3D images.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Statistical analyses\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eStatistical analysis was conducted using Origin Parametric software. Data is expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical analyses were performed using one-way ANOVA and Fisher\u0026rsquo;s Test. Statistical significance was defined as *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Construction of Mouse Engineered Neural Tissue (mENT) in Vitro\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eBuilding on our previous work demonstrating that 150 mV/mm physiological DC EFs induce neuronal differentiation in 3D Matrigel cultures(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), we generated EGFP-expressing mENT (EGFP-ENT) to track graft integration. EGFP-NSCs (characterized in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) were suspended in Matrigel and exposed to DC EFs (150 mV/mm, 30 min/day for 3 consecutive days), followed by 4 days of differentiation.\u003c/p\u003e\u003cp\u003e3D reconstruction imaging revealed that the EGFP-ENT constructs with consistent diameters (4\u0026thinsp;~\u0026thinsp;5 mm; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and thicknesses (180\u0026thinsp;~\u0026thinsp;300 \u0026micro;m; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and E). At 7 days post-differentiation, immunostaining showed predominant neuronal commitment, with 72.49\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0% of EGFP\u0026thinsp;+\u0026thinsp;cells expressing the early neuronal marker Tuj1, compared to only 8.94\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5% GFAP\u0026thinsp;+\u0026thinsp;astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). By day 14, neuronal maturation increased further, with 74.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.0% of cells expressing the mature marker MAP2, while GFAP\u0026thinsp;+\u0026thinsp;astrocytes declined to 6.21\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eWithin the 3D tissue, neurites formed elaborate networks (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) with abundant synaptophysin\u0026thinsp;+\u0026thinsp;puncta (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Transmission electron microscopy (TEM) confirmed ultrastructural hallmarks of functional neural tissue, including vesicle-rich presynaptic terminals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK), thickened pre- and postsynaptic densities (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI, J), and myelinated axons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH), indicating advanced neural tissue maturation.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Superior Structural Stability of 3D ENT in Large-volume lesions\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe experimental design and timeline are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. Sixty-six adult female C57BL/6 mice were randomized into four groups, including a group that received laminectomy only (sham-operated group, n\u0026thinsp;=\u0026thinsp;6), a SCI group that received normal saline (SCI group, n\u0026thinsp;=\u0026thinsp;20), a SCI group that received EGFP-ENT transplantation (ENT group, n\u0026thinsp;=\u0026thinsp;20), and a SCI group that received EGFP-NSC transplantation (NSC group, n\u0026thinsp;=\u0026thinsp;20). Histological analysis confirmed successful T9-11 hemisecting injuries, characterized by cavitation and inflammatory infiltration (Figure S2).\u003c/p\u003e\u003cp\u003eSmall-animal imaging at 24 hours post-transplantation verified graft placement, with both EGFP-ENT and EGFP-NSCs filling the lesion cavity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and C). By 7 days post-transplantation (dpt), the ENT group maintained dense EGFP\u0026thinsp;+\u0026thinsp;cell populations organized into intact tissue structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), whereas NSC grafts showed minimal EGFP\u0026thinsp;+\u0026thinsp;cell retention (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), demonstrating the superior structural stability of 3D ENT in large-volume lesions.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Transplantation of EGFP-ENTs Enhances Locomotor and Electrophysiological Recovery in SCI Mice\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eBMS assessments revealed progressive recovery across all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). While the SCI group reached a plateau at 4.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 by 28 days post-transplantation (dpt), the EGFP-ENT group achieved significantly higher scores (7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 vs EGFP-NSC: 5.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\u003cp\u003eGait analysis showed distinct recovery patterns: 1 day post-SCI (dpi): Complete hindlimb paralysis in all injured groups; 5 dpi: EGFP-ENT mice developed ankle movement (SCI: no volitional movement); 7 dpi: EGFP-ENT mice showed consistent foot-ground contact (SCI: only sporadic limb movement); 10\u0026thinsp;~\u0026thinsp;14 dpi: EGFP-ENT mice achieved weight-bearing (SCI: occasional standing with instability); 21\u0026thinsp;~\u0026thinsp;28 dpi: EGFP-ENT mice demonstrated coordinated plantar stepping (SCI: no coordinated movement) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, Video S1-4).\u003c/p\u003e\u003cp\u003eCortical somatosensory/motor evoked potentials (CSEP/CMEP) measurements showed: 24 h post-SCI: 80% reduction in amplitude (vs sham) and latency increased by 150% (vs sham). 28 dpt: ENT group: Near-normal amplitudes (90% of sham) and latencies (110% of sham); NSC group: Partial recovery (60% amplitude, 130% latency vs sham); SCI group: Minimal improvement (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-E).\u003c/p\u003e\u003cp\u003eThese results demonstrate that EGFP-ENT transplantation significantly enhances both structural and functional repair after SCI compared to single-cell suspensions.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Spatiotemporal Dynamics of EGFP-ENT Integration in Host Spinal Cord\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eWe systematically evaluated graft survival, migration, and synaptic integration at 7, 14, and 28 dpt using immunofluorescence for lineage-specific markers (GFAP, MAP2, MBP, ChAT and synaptophysin) and NSC marker Nestin. Migration distances were quantified from the lesion epicenter (n\u0026thinsp;=\u0026thinsp;3 mice/timepoint).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e\u003cb\u003e3.4.1 Short-Term Engraftment (7 dpt)\u003c/b\u003e\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eEGFP-ENT grafts showed EGFP-ENT filled into the lesion site (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), with cells migrating into host tissue. Migrating EGFP\u0026thinsp;+\u0026thinsp;cells exhibited dual differentiation: astrocytic lineage: GFAP\u0026thinsp;+\u0026thinsp;cells at graft-host interface (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB); neuronal lineage: MAP2\u0026thinsp;+\u0026thinsp;cells in lesion site and white matter (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Notably, no EGFP+/MAP2\u0026thinsp;+\u0026thinsp;cells were detected in gray matter (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), indicating stage-limited integration.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\u003ch2\u003e\u003cb\u003e3.4.2 Intermediate Phase (14 dpt)\u003c/b\u003e\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eMigration distance increased to 1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31 mm, with three distinct populations: central canal homing (EGFP+/MAP2- clusters) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C), white matter penetration (EGFP+/MAP2\u0026thinsp;+\u0026thinsp;neurons) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), gray matter entry (EGFP+/SYP- cells) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and F). It is worth noting that at this stage, the cells entering the grey matter have not yet expressed synaptophysin.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\u003ch2\u003e\u003cb\u003e3.4.3 Long-Term Integration (28 dpt)\u003c/b\u003e\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eOnly a few EGFP\u0026thinsp;+\u0026thinsp;cells remained at the graft core (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), maturated graft-derived EGFP+/MAP2\u0026thinsp;+\u0026thinsp;neurons exhibited in gray matter (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) and interacted with host neurons, which can be identified by the presenting of both EGFP+/SYP+ (yellow arrows) and EGFP-/SYP\u0026thinsp;+\u0026thinsp;cells (white arrows) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-F).\u003c/p\u003e\u003cp\u003eBy analyzing the ultrastructure of the spinal cord after injury and transplantation of ENT, we found that the spinal cord tissue at the injury site was structurally disordered, with vacuoles appearing within the cells, and the myelin sheath structure became thinner and incomplete (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH) compared to the normal spinal cord tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). On the contrary, in the individuals receiving ENT transplantation, the structure of the spinal cord injury center was relatively compact (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). Long protrusions were seen to be arranged in parallel (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ). At the injury site, intact myelin sheaths (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK) and larger blood vessels invading (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL) could be observed.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThese results indicate that bioelectric stimulation generates 3D ENT with pre-formed neuronal cells. After transplantation into spinal cord lesions, ENT-derived cells exhibit progressive migration and differentiation into mature neurons, and functional synaptic integration with host circuits, enabling motor recovery.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Niche-Directed Homing and Lineage Diversification of ENT-Derived Cells\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAt 14 dpt, spatial analysis revealed a striking compartmentalization of EGFP\u0026thinsp;+\u0026thinsp;cell fate determination (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), with three distinct microanatomical distributions. First, central canal homing guided by the stem cell niche engagement. NSC Retention: migrated EGFP\u0026thinsp;+\u0026thinsp;cells formed periventricular clusters, exhibiting strong Nestin co-expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Second, reconstruction of the motor circuit dominates the differentiation of spinal gray matter cells. Motor neuron specification: gray matter-infiltrating EGFP\u0026thinsp;+\u0026thinsp;cells expressed choline acetyltransferase (ChAT), displaying characteristic large somata and ventral horn localization (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), suggesting microenvironment-driven lineage bias. Third, oligodendrogenesis and myelination potential promotes white matter integration. We also detected MBP-positive engrafted cells in the spinal cord white matter (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), exhibiting mature, multi-lamellar sheaths (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK).\u003c/p\u003e\u003cp\u003eThese results implicated the unique characteristics of ENT transplantation: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Niche-specific recruitment: central canal homing mirrors endogenous NSC trafficking, implying conserved chemoattractant pathways; (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) functional diversification: simultaneous ChAT\u0026thinsp;+\u0026thinsp;neuron and MBP\u0026thinsp;+\u0026thinsp;oligodendrocyte generation demonstrates multi-lineage reparative potential.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Transcriptomic Mechanisms of EF-Induced Neuronal Differentiation\u003c/h2\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003e\u003cb\u003e3.6.1 RNA-Seq Profiling of EF-Stimulated 3D ENT\u003c/b\u003e\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTo elucidate the molecular basis of EF-enhanced neurogenesis, we performed RNA sequencing on 3D ENT cultures following 7 days of EF stimulation (150 mV/mm, 30 min/day) versus unstimulated controls (n\u0026thinsp;=\u0026thinsp;3 replicates/group). After stringent quality control (Q30\u0026thinsp;\u0026gt;\u0026thinsp;90%, Phred score\u0026thinsp;\u0026ge;\u0026thinsp;30), reads were aligned to GRCm38/mm10 using STAR (v2.7.10b), achieving 92.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1% mapping efficiency.\u003c/p\u003e\u003cp\u003eDifferential expression analysis revealed that there are 375 significantly regulated genes (adjusted p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) out of 12,661 detected transcripts (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA and B). Downregulated Genes (n\u0026thinsp;=\u0026thinsp;174) are enriched for gliogenic (Gfap \u0026darr;2.1-fold) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC).The upregulated Genes (n\u0026thinsp;=\u0026thinsp;201) are dominated by neurodevelopmental regulators (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD) like Nrgn (Neurogranin, calcium-calmodulin signaling modulator, \u0026uarr;8.2-fold, p\u0026thinsp;=\u0026thinsp;3.2\u0026times;10⁻⁷) and Camk2a (Synaptic differentiation mediator, \u0026uarr;5.6-fold, p\u0026thinsp;=\u0026thinsp;1.8\u0026times;10⁻⁵).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\u003ch2\u003e\u003cb\u003e3.6.2 Functional Annotation and Pathway Enrichment Analysis\u003c/b\u003e\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eClusterProfiler software performed GO functional enrichment analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA) and KEGG pathway enrichment analysis of the differential gene set (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). For results of GO functional enrichment, a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was used as the threshold for significant enrichment. ClusterProfiler identified three key functional clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA) including: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Biological Processes: synapse assembly (FDR\u0026thinsp;=\u0026thinsp;2.1\u0026times;10⁻⁷) and neuron projection development (FDR\u0026thinsp;=\u0026thinsp;4.3\u0026times;10⁻⁶) (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) Cellular Components: dendritic spine (FDR\u0026thinsp;=\u0026thinsp;7.8\u0026times;10⁻⁵) and presynaptic active zone (FDR\u0026thinsp;=\u0026thinsp;1.2\u0026times;10⁻⁴); (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) Molecular Functions: voltage-gated calcium channel activity (FDR\u0026thinsp;=\u0026thinsp;3.5\u0026times;10⁻\u0026sup3;) and tubulin binding (FDR\u0026thinsp;=\u0026thinsp;5.1\u0026times;10⁻\u0026sup3;).\u003c/p\u003e\u003cp\u003eKEGG pathway enrichment (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB) mainly included the following pathways (top enriched pathways, q\u0026thinsp;\u0026lt;\u0026thinsp;0.01): Calcium signaling (8 genes, q\u0026thinsp;=\u0026thinsp;2.4\u0026times;10⁻⁴), PI3K-AKT (7 genes, q\u0026thinsp;=\u0026thinsp;6.1\u0026times;10⁻⁴), Wnt (5 genes, q\u0026thinsp;=\u0026thinsp;8.3\u0026times;10⁻\u0026sup3;), Axon guidance (6 genes, q\u0026thinsp;=\u0026thinsp;9.7\u0026times;10⁻\u0026sup3;).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003e\u003cb\u003e3.6.3 Protein-Protein Interaction (PPI) Network\u003c/b\u003e\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFrom the 375 DEGs, PPI analysis (STRING DB) revealed that there are 367 nodes and 331 edges (interaction enrichment p\u0026thinsp;=\u0026thinsp;1.0\u0026times;10⁻\u0026sup1;⁶). Among the identified genes, we extracted and presented five hub genes that are closely associated with calcium ion channels and neural differentiation. (Refer to Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e): Camk2a (degree\u0026thinsp;=\u0026thinsp;28), Pik3r1 (PI3K regulatory subunit, degree\u0026thinsp;=\u0026thinsp;25), Cacna1a (voltage-gated calcium channel, degree\u0026thinsp;=\u0026thinsp;22), Dvl1 (Wnt signaling, degree\u0026thinsp;=\u0026thinsp;19) and Ntrk2 (BDNF receptor, degree\u0026thinsp;=\u0026thinsp;17).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eKey Mechanistic Insights\u003c/b\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eBased on the above analysis, we propose a potential mechanism of the regulation of neuronal differentiation by EFs. EF stimulation activates a calcium-PI3K/Wnt signaling axis that drives neuronal commitment while suppressing glial fate. Synaptogenic programs are initiated prior to transplantation, priming ENT for functional integration. Hub gene topology suggests EF mimics endogenous neurotrophic signaling (BDNF/NT-3 pathways).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eSpinal cord injury initiates a pathological cascade involving inflammatory infiltration, glial scarring, and cavitation, leading to axonal degeneration and functional impairment(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). While NSC transplantation has shown promise in SCI repair(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), its efficacy is limited in severe cases due to poor cell survival within large lesion cavities and low neuronal differentiation rate(\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) .\u003c/p\u003e\u003cp\u003eOur study addresses four critical barriers to repair: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Microenvironment modulation: 3D ENT structure mitigates inhibitory signals; (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) Cell replacement: pre-differentiated neurons bypass host differentiation blockade; (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) Circuit integration: pre-formed neurons and oligodendrocytes enable rapid host connectivity; (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) Niche-directed homing and lineage diversification of ENT-derived cells.\u003c/p\u003e\u003cp\u003eIn this study, the application of physiological EFs induced the pre-differentiation of NSCs in 3D matrix in vitro, resulting in the construction of mature neural tissues with a high proportion of neurons and a large number of synapses and myelin sheaths, forming a well-developed neural network within the ENT. This mature 3D neural tissue has a self-regulating microenvironment(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Thus, it can possess a certain ability to resist the inhibitory microenvironment of damaged spinal cord, facilitating its survival and continued growth in the host, and also greatly shortens the time from stem cells to neurons, reduces the risks of abnormal proliferation(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eUnlike previous NSC differentiation studies under 3D environment reporting\u0026thinsp;\u0026lt;\u0026thinsp;30% neuronal differentiation(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e), our ENT achieved\u0026thinsp;\u0026gt;\u0026thinsp;70% neurons, likely due to EF-induced PI3K /AKT/β-catenin activation, which suppresses glial fate(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOn the other hand, transplantation in the acute phase of injury was used not only to avoid excessive hyperplasia of glial scar and further deterioration of microenvironment after SCI, but also to reduce the damage to mice caused by multiple surgeries(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCompared to conventional NSC therapy(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e), our approach provides some advantages: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Structural protection: providing 3D structural support to reduce mechanical stress; (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) Pre-differentiation: pre-differentiating NSCs into neurons before transplantation, avoiding hostile microenvironment effects; (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) Network maturity: accelerating functional integration through pre-differentiated neurons and synaptic connections.\u003c/p\u003e\u003cp\u003eThe BMS score results showed that within 28 days, the functional recovery speed of mice transplanted with EGFP-ENT was faster than that of the group without EGFP-ENT transplantation, which also indicates that the transplantation of EGFP-ENT does indeed help the functional recovery of SCI mice.\u003c/p\u003e\u003cp\u003eNotably, we investigated spatiotemporal integration dynamics of ENT in SCI mice. 7 dpt: Radial dispersion with dual GFAP+/MAP2\u0026thinsp;+\u0026thinsp;differentiation; 14 dpt: Niche homing; 28 dpt: Functional synapses on ChAT\u0026thinsp;+\u0026thinsp;motor neurons. This homing behavior likely results from chemoattractants (e.g., SDF-1/CXCR4 axis(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e)) secreted by the canal microenvironment, suggesting ENT-derived NSCs may synergize with host stem cells to enhance repair. The homing of EGFP+/Nestin⁺ cells to the central canal\u0026mdash;a niche for endogenous NSCs\u0026mdash;suggests their potential to recruit host stem cells for repair(\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this study, not only showed NSCs homing behavior, but also mature neurons. At 28 dpt, we detected typical ChAT-positive motoneurons in the gray matter of the spinal cord, with large cell bodies, large round nuclei, well-developed processes, and numerous synaptophysin-positive sites around the cell bodies and processes. ChAT-positive neurons were motor neurons located in the anterior horn of the gray matter of the spinal cord, indicating that the neurons in EGFP-ENT migrate to the interior of the spinal cord, while continuing to differentiate and mature into different neuronal subtypes and migrate to specific locations to establish synaptic connections with different cells. This may be the structural basis for ENT transplantation to promote functional recovery in mice with SCI(\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWe further investigated the mechanistic insights of EF-regulated neuronal differentiation. Transcriptomics revealed EF-activated pathways which include Calcium-CAMK2A: drives axonal growth (\u0026uarr;8.2-fold Nrgn); PI3K-AKT: enhances survival (\u0026uarr;5.6-fold Pik3r1); Wnt/β-catenin: suppresses glial fate (Gfap \u0026darr;2.1-fold)(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn conclusion, we have developed a novel cell transplantation therapy that uses electrical stimulation to pre-build 3D neural tissues for the treatment of SCI. The therapeutic effect of this treatment is significantly better than that of NSC treatment. ENT transplantation promoted the recovery of the mice's motor function in the short term and established neural circuits (CSEP/CMEP significantly improved).\u003c/p\u003e\u003cp\u003eOne limitation of this study is that, compared with traditional spinal cord contusion or resection surgeries, we used an uncommon SCI model, which enabled us to transplant the corresponding volume of ENT and evaluate the histological changes in the spinal cord injury and the transplantation area. Additionally, avoiding the death of graft cells due to immune rejection reactions remains a major challenge after transplantation(\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). This study only observed the grafts for a short period (28 days), and the long-term survival of the graft cells requires the use of immunosuppressive drugs to solve this problem, which requires long-term observation in the future(\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOverall, although this study has certain limitations, electrical stimulation of pre-differentiated ENT transplantation will still become a new approach for treating SCI, and it may also provide new ideas and methods for tissue engineering transplantation to treat central nervous system injuries.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eOur ENT platform achieves structural and functional restoration through pre-formed neural networks; it realizes the synergy between the host and the graft through specialized and niche-adaptive homing mechanisms and provides clinically valuable targets (the calcium-PI3K-Wnt axis). The current limitations include temporal mismatch: there is a difference between the recovery time of mice (28 days) and that of humans (several months to several years)(\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e), and spatial scaling issues: a porcine model is needed to simulate the relevant distances in humans. Future work will focus on large animal validation and the production of ENT in accordance with GMP standards.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors declare that they have not use AI-generated work in this manuscript\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003eAnimal experiments were approved by the Ethics Committee of Experimental Animals in Basic Medical College of Jilin University ((2025) Study No. 411).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eAll authors have declared no conflict of interest in this manuscript.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by grants from Department of Science and Technology of Jilin Province: Key Scientific and Technological Research and Development Projects (No. 20180201026YY), to XTM.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eXTM and FW designed the study. XTM and XYY wrote the manuscript. RRL made the figures, carried out most experiments and interpreted results. XYY analyzed the transcriptome results. JYZ carried out animal experiments. XTM and FW conceptualized and supervised the experiments. ZYD, JZ and ZCW completed final editing and revision of the manuscript. All authors have given approval to the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eFigures were created with BioRender.com.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data that support the findings of this study are included within the article (and any supplementary files).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFu C, Jin X, Ji K, Lan K, Mao X, Huang Z, et al. Macrophage-targeted Mms6 mRNA-lipid nanoparticles promote locomotor functional recovery after traumatic spinal cord injury in mice. 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Stem Cells Transl Med. 2025;14(2).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKjell J, Olson L. Rat models of spinal cord injury: from pathology to potential therapies. Dis Model Mech. 2016;9(10):1125\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-regenerative-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"npjregenmed","sideBox":"Learn more about [npj Regenerative Medicine](http://www.nature.com/npjregenmed/)","snPcode":"41536","submissionUrl":"https://mts-npjregenmed.nature.com/cgi-bin/main.plex","title":"npj Regenerative Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Spinal cord injury, Neural stem cells, Tissue engineering, Bioelectric stimulation, Neuronal differentiation","lastPublishedDoi":"10.21203/rs.3.rs-7862539/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7862539/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSpinal cord injury (SCI) creates a prohibitive microenvironment that limits the efficacy of neural stem cell (NSC) therapies. We developed electrically preconditioned engineered neural tissues (ENT) to address these limitations through: (1) pre-establishment of functional neural networks in vitro, and (2) enhanced host integration capacity. EGFP-expressing NSCs were differentiated in 3D Matrigel under 150 mV/mm physiological electric fields (EFs) and transplanted into T10 hemisection SCI mice. Outcomes were assessed through: Basso Mouse Scale (BMS) scoring, multiplex immunofluorescence (Nestin/MAP2/GFAP/MBP/Synaptophysin/ChAT), cortical somatosensory/motor evoked potentials (CSEP/CMEP), RNA sequencing and pathway analysis. We conducted a comprehensive evaluations of the histological structure and function of EF-preconditioned ENT and the mice that received ENT transplantation: (1) in vitro maturation of ENT: high neuronal differentiation, dense synaptic networks and myelinated axon; (2) in vivo integration: niche-directed migration (graft-derived cells showed central canal (Nestin+ cells) and grey matter (ChAT+ cells) homing), achieved functional synaptic integration and correlated with motor recovery. Mechanistic analysis revealed EF activation of pro-neuronal pathways and gliogenesis suppression. These results demonstrate that EF-preconditioned ENT enables structural neural network reconstruction, niche-directed homing, functional synaptic integration and significant motor recovery.\u003c/p\u003e","manuscriptTitle":"Electrically Preconditioned Engineered Neural Tissues Promote Structural and Functional Repair in Spinal Cord Injury Through Niche-Directed Neural Circuit Reconstruction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-25 14:54:17","doi":"10.21203/rs.3.rs-7862539/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-28T17:55:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-19T17:05:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-06T16:47:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"103693801705695571067331098065052412562","date":"2025-11-26T13:33:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"184968917498906829036851889248733551274","date":"2025-11-14T09:34:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-14T09:19:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-14T01:40:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Regenerative Medicine","date":"2025-11-14T01:36:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-regenerative-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"npjregenmed","sideBox":"Learn more about [npj Regenerative Medicine](http://www.nature.com/npjregenmed/)","snPcode":"41536","submissionUrl":"https://mts-npjregenmed.nature.com/cgi-bin/main.plex","title":"npj Regenerative Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dfcad77f-be22-4839-87f5-ccb9116f93ac","owner":[],"postedDate":"November 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":58492833,"name":"Biological sciences/Neuroscience"},{"id":58492834,"name":"Biological sciences/Stem cells"}],"tags":[],"updatedAt":"2026-04-29T16:08:32+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-25 14:54:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7862539","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7862539","identity":"rs-7862539","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}