Effects of tail nerve electrical stimulation on the activation and plasticity of the lumbar locomotor circuits and the prevention of skeletal muscle atrophy after spinal cord transection in rats.

INTRODUCTION: Severe spinal cord injury results in the loss of neurons in the relatively intact spinal cord below the injury area and skeletal muscle atrophy in the paralyzed limbs. These pathological processes are significant obstacles for motor function reconstruction. OBJECTIVE: We performed tail nerve electrical stimulation (TNES) to activate the motor neural circuits below the injury site of the spinal cord to elucidate the regulatory mechanisms of the excitatory afferent neurons in promoting the reconstruction of locomotor function. METHODS: Eight days after T10 spinal cord transection in rats, TNES was performed for 7 weeks. Behavioral scores were assessed weekly. Electrophysiological tests and double retrograde tracings were performed at week 8. RESULTS: After 7 weeks of TNES treatment, there was restoration in innervation, the number of stem cells, and mitochondrial metabolism in the rats' hindlimb muscles. Double retrograde tracings of the tail nerve and sciatic nerve further confirmed the presence of synaptic connections between the tail nerve and central pattern generator (CPG) neurons in the lumbar spinal cord, as well as motor neurons innervating the hindlimb muscles. CONCLUSION: The mechanisms of TNES induced by the stimulation of primary afferent nerve fibers involves efficient activation of the motor neural circuits in the lumbosacral segment, alterations of synaptic plasticity, and the improvement of muscle and nerve regeneration, which provides the structural and functional foundation for the future use of cutting-edge biological treatment strategies to restore voluntary movement of paralyzed hindlimbs.

The therapeutic mechanism of transcranial iTBS on nerve regeneration and functional recovery in rats with complete spinal cord transection.

Background: After spinal cord transection injury, the inflammatory microenvironment formed at the injury site, and the cascade of effects generated by secondary injury, results in limited regeneration of injured axons and the apoptosis of neurons in the sensorimotor cortex (SMC). It is crucial to reverse these adverse processes for the recovery of voluntary movement. The mechanism of transcranial intermittent theta-burst stimulation (iTBS) as a new non-invasive neural regulation paradigm in promoting axonal regeneration and motor function repair was explored by means of a severe spinal cord transection. Methods: Rats underwent spinal cord transection and 2 mm resection of spinal cord at T10 level. Four groups were studied: Normal (no lesion), Control (lesion with no treatment), sham iTBS (lesion and no functional treatment) and experimental, exposed to transcranial iTBS, 72 h after spinal lesion. Each rat received treatment once a day for 5 days a week; behavioral tests were administered one a week. Inflammation, neuronal apoptosis, neuroprotective effects, regeneration and synaptic plasticity after spinal cord injury (SCI) were determined by immunofluorescence staining, western blotting and mRNA sequencing. For each rat, anterograde tracings were acquired from the SMC or the long descending propriospinal neurons and tested for cortical motor evoked potentials (CMEPs). Regeneration of the corticospinal tract (CST) and 5-hydroxytryptamine (5-HT) nerve fibers were analyzed 10 weeks after SCI. Results: When compared to the Control group, the iTBS group showed a reduced inflammatory response and reduced levels of neuronal apoptosis in the SMC when tested 2 weeks after treatment. Four weeks after SCI, the neuroimmune microenvironment at the injury site had improved in the iTBS group, and neuroprotective effects were evident, including the promotion of axonal regeneration and synaptic plasticity. After 8 weeks of iTBS treatment, there was a significant increase in CST regeneration in the region rostral to the site of injury. Furthermore, there was a significant increase in the number of 5-HT nerve fibers at the center of the injury site and the long descending propriospinal tract (LDPT) fibers in the region caudal to the site of injury. Moreover, CMEPs and hindlimb motor function were significantly improved. Conclusion: Neuronal activation and neural tracing further verified that iTBS had the potential to provide neuroprotective effects during the early stages of SCI and induce regeneration effects related to the descending motor pathways (CST, 5-HT and LDPT). Furthermore, our results revealed key relationships between neural pathway activation, neuroimmune regulation, neuroprotection and axonal regeneration, as well as the interaction network of key genes.

A biocompatible gelatin sponge scaffold confers robust tissue remodeling after spinal cord injury in a non-human primate model.

We previously constructed a three-dimensional gelatin sponge (3D-GS) scaffold as a delivery vehicle for therapeutic cells and trophic factors in the treatment of spinal cord injury (SCI), and this study aimed to assess the biosafety and efficacy of the scaffold in a non-human primate SCI model. However, because it has only been tested in rodent and canine models, the biosafety and efficacy of the scaffold should ideally be assessed in a non-human primate SCI model before its use in the clinic. No adverse reactions were observed over 8 weeks following 3D-GS scaffold implantation into in a Macaca fascicularis with hemisected SCI. Scaffold implantation also did not add to neuroinflammatory or astroglial responses already present at the injured site, suggesting good biocompatibility. Notably, there was a significant reduction in α-smooth muscle actin (αSMA)-positive cells at the injury/implantation interface, leading to alleviation of fibrotic compression of the residual spinal cord tissue. The regenerating tissue in the scaffold showed numerous cells migrating into the implant secreting abundant extracellular matrix, resulting in a pro-regenerative microenvironment. Consequently, nerve fiber regeneration, myelination, vascularization, neurogenesis, and electrophysiological improvements were achieved. These results indicated that the 3D-GS scaffold had good histocompatibility and effectiveness in the structural repair of injured spinal cord tissue in a non-human primate and is suitable for use in the treatment of patients with SCI.

Tail nerve electrical stimulation promoted the efficiency of transplanted spinal cord-like tissue as a neuronal relay to repair the motor function of rats with transected spinal cord injury.

Following transected spinal cord injury (SCI), there is a critical need to restore nerve conduction at the injury site and activate the silent neural circuits caudal to the injury to promote the recovery of voluntary movement. In this study, we generated a rat model of SCI, constructed neural stem cell (NSC)-derived spinal cord-like tissue (SCLT), and evaluated its ability to replace injured spinal cord and repair nerve conduction in the spinal cord as a neuronal relay. The lumbosacral spinal cord was further activated with tail nerve electrical stimulation (TNES) as a synergistic electrical stimulation to better receive the neural information transmitted by the SCLT. Next, we investigated the neuromodulatory mechanism underlying the action of TNES and its synergism with SCLT in SCI repair. TNES promoted the regeneration and remyelination of axons and increased the proportion of glutamatergic neurons in SCLT to transmit brain-derived neural information more efficiently to the caudal spinal cord. TNES also increased the innervation of motor neurons to hindlimb muscle and improved the microenvironment of muscle tissue, resulting in effective prevention of hindlimb muscle atrophy and enhanced muscle mitochondrial energy metabolism. Tracing of the neural circuits of the sciatic nerve and tail nerve identified the mechanisms responsible for the synergistic effects of SCLT transplantation and TNES in activating central pattern generator (CPG) neural circuits and promoting voluntary motor function recovery in rats. The combination of SCLT and TNES is expected to provide a new breakthrough for patients with SCI to restore voluntary movement and control their muscles.

Multimodal Repair of Spinal Cord Injury With Mesenchymal Stem Cells.

Spinal cord injury (SCI) is a result of a devastating injury to the central nervous system. Currently, there is no effective treatment available for these patients. The possible use of mesenchymal stem cell (MSC)-based treatment for SCI has been the focus of extensive investigations and is increasingly moving from the bench to bedside. Both experimental observations and clinical studies have shown the safety and efficacy of MSCs in managing SCI. However, the exact mechanism by which MSCs contribute to the repair of the injured spinal cord remains to be elucidated. In this review, we aim to summarize current research findings about the role of MSCs in improving complex pathology after SCI. MSCs exert a multimodal repair mechanism targeting multiple events in the secondary injury cascade. Our recent results showing the perineurium-like differentiation of surviving MSCs in the injured spinal cord may further the understanding of the fate of transplanted MSCs. These findings provide fundamental support for the clinical use of MSCs in SCI patients. Under experimental conditions, combining novel physical, chemical, and biological approaches led to significant improvements in the therapeutic efficacy of MSCs. These findings hold promise for the future of cell-based clinical treatment of SCI.

Electro-acupuncture and its combination with adult stem cell transplantation for spinal cord injury treatment: A summary of current laboratory findings and a review of literature.

The incidence and disability rate of spinal cord injury (SCI) worldwide are high, imposing a heavy burden on patients. Considerable research efforts have been directed toward identifying new strategies to effectively treat SCI. Governor Vessel electro-acupuncture (GV-EA), used in traditional Chinese medicine, combines acupuncture with modern electrical stimulation. It has been shown to improve the microenvironment of injured spinal cord (SC) by increasing levels of endogenous neurotrophic factors and reducing inflammation, thereby protecting injured neurons and promoting myelination. In addition, axons extending from transplanted stem cell-derived neurons can potentially bridge the two severed ends of tissues in a transected SC to rebuild neuronal circuits and restore motor and sensory functions. However, every single treatment approach to severe SCI has proven unsatisfactory. Combining different treatments-for example, electro-acupuncture (EA) with adult stem cell transplantation-appears to be a more promising strategy. In this review, we have summarized the recent progress over the past two decades by our team especially in the use of GV-EA for the repair of SCI. By this strategy, we have shown that EA can stimulate the nerve endings of the meningeal branch. This would elicit the dorsal root ganglion neurons to secrete excess amounts of calcitonin gene-related peptide centrally in the SC. The neuropeptide then activates the local cells to secrete neurotrophin-3 (NT-3), which mediates the survival and differentiation of donor stem cells overexpressing the NT-3 receptor, at the injury/graft site of the SC. Increased local production of NT-3 facilitates reconstruction of host neural tissue such as nerve fiber regeneration and myelination. All this events in sequence would ultimately strengthen the cortical motor-evoked potentials and restore the motor function of paralyzed limbs. The information presented herein provides a basis for future studies on the clinical application of GV-EA and adult stem cell transplantation for the treatment of SCI.

Transcription Profiling of a Revealed the Potential Molecular Mechanism of Governor Vessel Electroacupuncture for Spinal Cord Injury in Rats.

OBJECTIVE: This study aimed to identify differentially expressed genes (DEGs) by transcriptome analysis to elucidate a potential mechanism by which governor vessel electroacupuncture (GV-EA) promotes neuronal survival, axonal regeneration, and functional recovery after complete transection spinal cord injury (SCI). METHODS: Sham, control, or GV-EA group adult female Sprague Dawley rats underwent a complete transection SCI protocol. SCI area RNA-seq investigated the DEGs of coding and noncoding RNAs 7 days post-SCI. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were used to classify DEGs functions, to explain a possible molecular mechanism. Immunofluorescence and BBB (Basso, Beattie, and Bresnahan) score were used to verify a GV-EA treatment effect following SCI. RESULTS: GV-EA treatment could regulate the expression of 173 mRNA, 260 lncRNA, and 153 circRNA genes among these DEGs resulted by SCI. GO enrichment analysis showed that the DEGs were most enriched in membrane, actin binding, and regulation of Toll-like receptor signaling pathway. KEGG pathway analysis showed enriched pathways (e.g. , Toll-like receptors, MAPK, Hippo signaling). According to the ceRNA network, miR-144-3p played a regulatory role by interacting with lncRNA and circRNA. GV-EA also promoted the injured spinal cord neuron survival, axonal regeneration, and functional improvement of hind limb locomotion. CONCLUSION: Results of our RNA-seq suggest that post-SCI GV-EA may regulate characteristic changes in transcriptome gene expression, potential critical genes, and signaling pathways, providing clear directions for further investigation into the mechanism of GV-EA in subacute SCI treatment. Moreover, we found that GV-EA promotes neuronal survival, nerve fiber extension, and motor function recovery in subacute SCI.

Construction of a niche-specific spinal white matter-like tissue to promote directional axon regeneration and myelination for rat spinal cord injury repair.

Directional axon regeneration and remyelination are crucial for repair of spinal cord injury (SCI), but existing treatments do not effectively promote those processes. Here, we propose a strategy for construction of niche-specific spinal white matter-like tissue (WMLT) using decellularized optic nerve (DON) loaded with neurotrophin-3 (NT-3)-overexpressing oligodendrocyte precursor cells. A rat model with a white matter defect in the dorsal spinal cord of the T10 segment was used. The WMLT transplantation group showed significant improvement in coordinated motor functions compared with the control groups. WMLT transplants integrated well with host spinal cord white matter, effectively addressing several barriers to directional axonal regeneration and myelination during SCI repair. In WMLT, laminin was found to promote development of oligodendroglial lineage (OL) cells by binding to laminin receptors. Interestingly, laminin could also guide linear axon regeneration via interactions with specific integrins on the axon surface. The WMLT developed here utilizes the unique microstructure and bioactive matrix of DON to create a niche rich in laminin, NT-3 and OL cells to achieve significant structural repair of SCI. Our protocol can help to promote research on repair of nerve injury and construction of neural tissues and organoids that form specific cell niches.

Developing a mechanically matched decellularized spinal cord scaffold for the in situ matrix-based neural repair of spinal cord injury.

Tissue engineering is a promising strategy to repair spinal cord injury (SCI). However, a bioscaffold with mechanical properties that match those of the pathological spinal cord tissue and a pro-regenerative matrix that allows robust neurogenesis for overcoming post-SCI scar formation has yet to be developed. Here, we report that a mechanically enhanced decellularized spinal cord (DSC) scaffold with a thin poly (lactic-co-glycolic acid) (PLGA) outer shell may fulfill the requirements for effective in situ neuroengineering after SCI. Using chemical extraction and electrospinning methods, we successfully constructed PLGA thin shell-ensheathed DSC scaffolds (PLGA-DSC scaffolds) in a way that removed major inhibitory components while preserving the permissive matrix. The DSCs exhibited good cytocompatibility with neural stem cells (NSCs) and significantly enhanced their differentiation toward neurons in vitro. Due to the mechanical reinforcement, the implanted PLGA-DSC scaffolds showed markedly increased resilience to infiltration by myofibroblasts and the deposition of dense collagen matrix, thereby creating a neurogenic niche favorable for the targeted migration, residence and neuronal differentiation of endogenous NSCs after SCI. Furthermore, PLGA-DSC presented a mild immunogenic property but prominent ability to polarize macrophages from the M1 phenotype to the M2 phenotype, leading to significant tissue regeneration and functional restoration after SCI. Taken together, the results demonstrate that the mechanically matched PLGA-DSC scaffolds show promise for effective tissue repair after SCI.

Decellularized optic nerve functional scaffold transplant facilitates directional axon regeneration and remyelination in the injured white matter of the rat spinal cord.

Axon regeneration and remyelination of the damaged region is the most common repair strategy for spinal cord injury. However, achieving good outcome remains difficult. Our previous study showed that porcine decellularized optic nerve better mimics the extracellular matrix of the embryonic porcine optic nerve and promotes the directional growth of dorsal root ganglion neurites. However, it has not been reported whether this material promotes axonal regeneration in vivo. In the present study, a porcine decellularized optic nerve was seeded with neurotrophin-3-overexpressing Schwann cells. This functional scaffold promoted the directional growth and remyelination of regenerating axons. In vitro, the porcine decellularized optic nerve contained many straight, longitudinal channels with a uniform distribution, and microscopic pores were present in the channel wall. The spatial micro topological structure and extracellular matrix were conducive to the adhesion, survival and migration of neural stem cells. The scaffold promoted the directional growth of dorsal root ganglion neurites, and showed strong potential for myelin regeneration. Furthermore, we transplanted the porcine decellularized optic nerve containing neurotrophin-3-overexpressing Schwann cells in a rat model of T10 spinal cord defect in vivo. Four weeks later, the regenerating axons grew straight, the myelin sheath in the injured/transplanted area recovered its structure, and simultaneously, the number of inflammatory cells and the expression of chondroitin sulfate proteoglycans were reduced. Together, these findings suggest that porcine decellularized optic nerve loaded with Schwann cells overexpressing neurotrophin-3 promotes the directional growth of regenerating spinal cord axons as well as myelin regeneration. All procedures involving animals were conducted in accordance with the ethical standards of the Institutional Animal Care and Use Committee of Sun Yat-sen University (approval No. SYSU-IACUC-2019-B034) on February 28, 2019.

Electroacupuncture facilitates the integration of a grafted TrkC-modified mesenchymal stem cell-derived neural network into transected spinal cord in rats via increasing neurotrophin-3.

AIMS: This study was aimed to investigate whether electroacupuncture (EA) would increase the secretion of neurotrophin-3 (NT-3) from injured spinal cord tissue, and, if so, whether the increased NT-3 would promote the survival, differentiation, and migration of grafted tyrosine kinase C (TrkC)-modified mesenchymal stem cell (MSC)-derived neural network cells. We next sought to determine if the latter would integrate with the host spinal cord neural circuit to improve the neurological function of injured spinal cord. METHODS: After NT-3-modified Schwann cells (SCs) and TrkC-modified MSCs were co-cultured in a gelatin sponge scaffold for 14 days, the MSCs differentiated into neuron-like cells that formed a MSC-derived neural network (MN) implant. On this basis, we combined the MN implantation with EA in a rat model of spinal cord injury (SCI) and performed immunohistochemical staining, neural tracing, electrophysiology, and behavioral testing after 8 weeks. RESULTS: Electroacupuncture application enhanced the production of endogenous NT-3 in damaged spinal cord tissues. The increase in local NT-3 production promoted the survival, migration, and maintenance of the grafted MN, which expressed NT-3 high-affinity TrkC. The combination of MN implantation and EA application improved cortical motor-evoked potential relay and facilitated the locomotor performance of the paralyzed hindlimb compared with those of controls. These results suggest that the MN was better integrated into the host spinal cord neural network after EA treatment compared with control treatment. CONCLUSIONS: Electroacupuncture as an adjuvant therapy for TrkC-modified MSC-derived MN, acted by increasing the local production of NT-3, which accelerated neural network reconstruction and restoration of spinal cord function following SCI.

An NT-3-releasing bioscaffold supports the formation of TrkC-modified neural stem cell-derived neural network tissue with efficacy in repairing spinal cord injury.

The mechanism underlying neurogenesis during embryonic spinal cord development involves a specific ligand/receptor interaction, which may be help guide neuroengineering to boost stem cell-based neural regeneration for the structural and functional repair of spinal cord injury. Herein, we hypothesized that supplying spinal cord defects with an exogenous neural network in the NT-3/fibroin-coated gelatin sponge (NF-GS) scaffold might improve tissue repair efficacy. To test this, we engineered tropomyosin receptor kinase C (TrkC)-modified neural stem cell (NSC)-derived neural network tissue with robust viability within an NF-GS scaffold. When NSCs were genetically modified to overexpress TrkC, the NT-3 receptor, a functional neuronal population dominated the neural network tissue. The pro-regenerative niche allowed the long-term survival and phenotypic maintenance of the donor neural network tissue for up to 8 weeks in the injured spinal cord. Additionally, host nerve fibers regenerated into the graft, making synaptic connections with the donor neurons. Accordingly, motor function recovery was significantly improved in rats with spinal cord injury (SCI) that received TrkC-modified NSC-derived neural network tissue transplantation. Together, the results suggested that transplantation of the neural network tissue formed in the 3D bioactive scaffold may represent a valuable approach to study and develop therapies for SCI.

Decellularization optimizes the inhibitory microenvironment of the optic nerve to support neurite growth.

Allogeneic or homologous tissue transplantation is an effective strategy to repair tissue injury. However, the central nervous tissues like the brain, spinal cord, and optic nerve are not ideal materials for nervous tissue regeneration due to the excessive axonal inhibitor cues in their microenvironments. In the present study, we found that decellularization optimizes the function of the adult optic nerve in supporting the oriented outgrowth of dorsal root ganglion (DRG) neurites. The neurites growing on the decellularized optic nerve (DON) showed longer extension distances than those growing on the normal optic nerve (ON). Neurite branching was also significantly increased on the DON compared to on the ON. Decellularization selectively removed some axon-inhibitory molecules such as myelin-associated glycoprotein (basically not detected in DON) and chondroitin sulfate proteoglycans (detected in DON at a level less than 0.3 fold that in ON) and preserved some axon-promoted extracellular matrix (ECM) proteins, including collagen IV and laminin (detected at levels 6.0-fold higher in DON than in ON). Furthermore, collagen IV and laminin were shown to be preserved in DON, and their binding activities with integrin α1 were retained to promote the extension of DRG neurites. Together, the findings provide a feasible way to optimize the axon-inhibited microenvironment of central nervous tissues and establish a theoretical basis for the application of DON scaffolds in repairing central nervous injury.

Tissue-Engineered Neural Network Graft Relays Excitatory Signal in the Completely Transected Canine Spinal Cord.

Tissue engineering produces constructs with defined functions for the targeted treatment of damaged tissue. A complete spinal cord injury (SCI) model is generated in canines to test whether in vitro constructed neural network (NN) tissues can relay the excitatory signal across the lesion gap to the caudal spinal cord. Established protocols are used to construct neural stem cell (NSC)-derived NN tissue characterized by a predominantly neuronal population with robust trans-synaptic activities and myelination. The NN tissue is implanted into the gap immediately following complete transection SCI of canines at the T10 spinal cord segment. The data show significant motor recovery of paralyzed pelvic limbs, as evaluated by Olby scoring and cortical motor evoked potential (CMEP) detection. The NN tissue survives in the lesion area with neuronal phenotype maintenance, improves descending and ascending nerve fiber regeneration, and synaptic integration with host neural circuits that allow it to serve as a neuronal relay to transmit excitatory electrical signal across the injured area to the caudal spinal cord. These results suggest that tissue-engineered NN grafts can relay the excitatory signal in the completely transected canine spinal cord, providing a promising strategy for SCI treatment in large animals, including humans.

Recovery of paralyzed limb motor function in canine with complete spinal cord injury following implantation of MSC-derived neural network tissue.

We have reported previously that bone marrow mesenchymal stem cell (MSC)-derived neural network scaffold not only survived in the injury/graft site of spinal cord but also served as a "neuronal relay" that was capable of improving the limb motor function in a complete spinal cord injury (SCI) rat model. It remained to be explored whether such a strategy was effective for repairing the large spinal cord tissue loss as well as restoring motor function in larger animals. We have therefore extended in this study to construct a canine MSC-derived neural network tissue in vitro with the aim to evaluate its efficacy in treating adult beagle dog subjected to a complete transection of the spinal cord. The results showed that after co-culturing with neurotropin-3 overexpressing Schwann cells in a gelatin sponge scaffold for 14 days, TrkC overexpressing MSCs differentiated into neuron-like cells. In the latter, some cells appeared to make contacts with each other through synapse-like structures with trans-synaptic electrical activities. Remarkably, the SCI canines receiving the transplantation of the MSC-derived neural network tissue demonstrated a gradual restoration of paralyzed limb motor function, along with improved electrophysiological presentation when compared with the control group. Magnetic resonance imaging and diffusion tensor imaging showed that the canines receiving the MSC-derived neural network tissue exhibited robust nerve tract regeneration in the injury/graft site. Histological analysis showed that some of the MSC-derived neuron-like cells had survived in the injury/graft site up to 6.5 months. Implantation of MSC-derived neural network tissue significantly improved the microenvironment of the injury/graft site. It is noteworthy that a variable number of them had integrated with the regenerating corticospinal tract nerve fibers and 5-HT nerve fibers through formation of synapse-like contacts. The results suggest that the transplanted MSC-derived neural network tissue may serve as a structural and functional "neuronal relay" to restore the paralyzed limb motor function in the canine with complete SCI.

Perineurium-like sheath derived from long-term surviving mesenchymal stem cells confers nerve protection to the injured spinal cord.

The functional multipotency enables mesenchymal stem cells (MSCs) promising translational potentials in treating spinal cord injury (SCI). Yet the fate of MSCs grafted into the injured spinal cord has not been fully elucidated even in preclinical studies, rendering concerns of their safety and genuine efficacy. Here we used a rat spinal cord transection model to evaluate the cell fate of allograft bone marrow derived MSCs. With the application of immunosuppressant, donor cells, delivered by biocompatible scaffold, survived up to 8 weeks post-grafting. Discernible tubes formed by MSCs were observed beginning 2 weeks after transplantation and they dominated the morphological features of implanted MSCs at 8 weeks post-grafting. The results of immunocytochemistry and transmission electron microscopy displayed the formation of perineurium-like sheath by donor cells, which, in a manner comparable to the perineurium in peripheral nerve, enwrapped host myelins and axons. The MSC-derived perineurium-like sheath secreted a group of trophic factors and permissive extracellular matrix, and served as a physical and chemical barrier to insulate the inner nerve fibers from ambient oxidative insults by the secretion of soluble antioxidant, superoxide dismutase-3 (SOD3). As a result, many intact regenerating axons were preserved in the injury/graft site following the forming of perineurium-like sheath. A parallel study utilizing a good manufacturing practice (GMP) grade human umbilical cord-derived MSCs or allogenic MSCs in an acute contusive/compressive SCI model exhibited a similar perineurium-like sheath formed by surviving donor cells in rat spinal cord at 3 weeks post-grafting. The present study for the first time provides an unambiguous morphological evidence of perineurium-like sheath formed by transplanted MSCs and a novel therapeutic mechanism of MSCs in treating SCI.

Autocrine fibronectin from differentiating mesenchymal stem cells induces the neurite elongation in vitro and promotes nerve fiber regeneration in transected spinal cord injury.

Extracellular matrix (ECM) expression is temporally and spatially regulated during the development of stem cells. We reported previously that fibronectin (FN) secreted by bone marrow mesenchymal stem cells (MSCs) was deposited on the surface of gelatin sponge (GS) soon after culture. In this study, we aimed to assess the function of accumulated FN on neuronal differentiating MSCs as induced by Schwann cells (SCs) in three dimensional transwell co-culture system. The expression pattern and amount of FN of differentiating MSCs was examined by immunofluorescence, Western blot and immunoelectron microscopy. The results showed that FN accumulated inside GS scaffold, although its mRNA expression in MSCs was progressively decreased during neural induction. MSC-derived neuron-like cells showed spindle-shaped cell body and long extending processes on FN-decorated scaffold surface. However, after blocking of FN function by application of monoclonal antibodies, neuron-like cells showed flattened cell body with short and thick neurites, together with decreased expression of integrin ß1. In vivo transplantation study revealed that autocrine FN significantly facilitated endogenous nerve fiber regeneration in spinal cord transection model. Taken together, the present results showed that FN secreted by MSCs in the early stage accumulated on the GS scaffold and promoted the neurite elongation of neuronal differentiating MSCs as well as nerve fiber regeneration after spinal cord injury. This suggests that autocrine FN has a dynamic influence on MSCs in a three dimensional culture system and its potential application for treatment of traumatic spinal cord injury. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1902-1911, 2016.

Transplantation of tissue engineering neural network and formation of neuronal relay into the transected rat spinal cord.

Severe spinal cord injury (SCI) causes loss of neural connectivity and permanent functional deficits. Re-establishment of new neuronal relay circuits after SCI is therefore of paramount importance. The present study tested our hypothesis if co-culture of neurotrophin-3 (NT-3) gene-modified Schwann cells (SCs, NT-3-SCs) and TrkC (NT-3 receptor) gene-modified neural stem cells (NSCs, TrkC-NSCs) in a gelatin sponge scaffold could construct a tissue engineering neural network for re-establishing an anatomical neuronal relay after rat spinal cord transection. Eight weeks after transplantation, the neural network created a favorable microenvironment for axonal regeneration and for survival and synaptogenesis of NSC-derived neurons. Biotin conjugates of cholera toxin B subunit (b-CTB, a transneuronal tracer) was injected into the crushed sciatic nerve to label spinal cord neurons. Remarkably, not only ascending and descending nerve fibers, but also propriospinal neurons, made contacts with b-CTB positive NSC-derived neurons. Moreover, b-CTB positive NSC-derived neurons extended their axons making contacts with the motor neurons located in areas caudal to the injury/graft site of spinal cord. Further study showed that NT-3/TrkC interactions activated the PI3K/AKT/mTOR pathway and PI3K/AKT/CREB pathway affecting synaptogenesis of NSC-derived neurons. Together, our findings suggest that NT-3-mediated TrkC signaling plays an essential role in constructing a tissue engineering neural network thus representing a promising avenue for effective exogenous neuronal relay-based treatment for SCI.

Graft of the gelatin sponge scaffold containing genetically-modified neural stem cells promotes cell differentiation, axon regeneration, and functional recovery in rat with spinal cord transection.

Biological materials combined with genetically-modified neural stem cells (NSCs) are candidate therapy targeting spinal cord injury (SCI). Based on our previous studies, here we performed gelatin sponge (GS) scaffold seeded with neurotrophin-3 (NT-3) and its receptor TrkC gene modifying NSCs for repairing SCI. Eight weeks later, compared with other groups, neurofilament-200 and 5-hydroxytryptamine positive nerve fibers were more in the injury site of the N+T-NSCs group. Immunofluorescence staining showed the grafted NSCs could differentiate into microtubule associated protein (Map2), postsynaptic density (PSD95), and mouse oligodendrocyte special protein (MOSP) positive cells. The percentage of the Map2, PSD95, and MOSP positive cells in the N+T-NSCs group was higher than the other groups. Immuno-electron microscopy showed the grafted NSCs making contact with each other in the injury site. Behavioral analysis indicated the recovery of hindlimbs locomotion was better in the groups receiving cell transplant, the best recovery was found in the N+T-NSCs group. Electrophysiology revealed the amplitude of cortical motor evoked potentials was increased significantly in the N+T-NSCs group, but the latency remained long. These findings suggest the GS scaffold containing genetically-modified NSCs may bridge the injury site, promote axon regeneration and partial functional recovery in SCI rats.

Integration of donor mesenchymal stem cell-derived neuron-like cells into host neural network after rat spinal cord transection.

Functional deficits following spinal cord injury (SCI) primarily attribute to loss of neural connectivity. We therefore tested if novel tissue engineering approaches could enable neural network repair that facilitates functional recovery after spinal cord transection (SCT). Rat bone marrow-derived mesenchymal stem cells (MSCs), genetically engineered to overexpress TrkC, receptor of neurotrophin-3 (NT-3), were pre-differentiated into cells carrying neuronal features via co-culture with NT-3 overproducing Schwann cells in 3-dimensional gelatin sponge (GS) scaffold for 14 days in vitro. Intra-GS formation of MSC assemblies emulating neural network (MSC-GS) were verified morphologically via electron microscopy (EM) and functionally by whole-cell patch clamp recording of spontaneous post-synaptic currents. The differentiated MSCs still partially maintained prototypic property with the expression of some mesodermal cytokines. MSC-GS or GS was then grafted acutely into a 2 mm-wide transection gap in the T9-T10 spinal cord segments of adult rats. Eight weeks later, hindlimb function of the MSC-GS-treated SCT rats was significantly improved relative to controls receiving the GS or lesion only as indicated by BBB score. The MSC-GS transplantation also significantly recovered cortical motor evoked potential (CMEP). Histologically, MSC-derived neuron-like cells maintained their synapse-like structures in vivo; they additionally formed similar connections with host neurites (i.e., mostly serotonergic fibers plus a few corticospinal axons; validated by double-labeled immuno-EM). Moreover, motor cortex electrical stimulation triggered c-fos expression in the grafted and lumbar spinal cord cells of the treated rats only. Our data suggest that MSC-derived neuron-like cells resulting from NT-3-TrkC-induced differentiation can partially integrate into transected spinal cord and this strategy should be further investigated for reconstructing disrupted neural circuits.