Stem cell-derived neuronal relay strategies and functional electrical stimulation for treatment of spinal cord injury.

The inability of adult mammals to recover function lost after severe spinal cord injury (SCI) has been known for millennia and is mainly attributed to a failure of brain-derived nerve fiber regeneration across the lesion. Potential approaches to re-establishing locomotor function rely on neuronal relays to reconnect the segregated neural networks of the spinal cord. Intense research over the past 30 years has focused on endogenous and exogenous neuronal relays, but progress has been slow and the results often controversial. Treatments with stem cell-derived neuronal relays alone or together with functional electrical stimulation offer the possibility of improved repair of neuronal networks. In this review, we focus on approaches to recovery of motor function in paralyzed patients after severe SCI based on novel therapies such as implantation of stem cell-derived neuronal relays and functional electrical stimulation. Recent research progress offers hope that SCI patients will one day be able to recover motor function and sensory perception.

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.

Electroacupuncture Facilitates the Integration of Neural Stem Cell-Derived Neural Network with Transected Rat Spinal Cord.

The hostile environment of an injured spinal cord makes it challenging to achieve higher viability in a grafted tissue-engineered neural network used to reconstruct the spinal cord circuit. Here, we investigate whether cell survival and synaptic transmission within an NT-3 and TRKC gene-overexpressing neural stem cell-derived neural network scaffold (NN) transplanted into transected spinal cord could be promoted by electroacupuncture (EA) through improving the microenvironment. Our results showed that EA facilitated the cell survival, neuronal differentiation, and synapse formation of a transplanted NN. Pseudorabies virus tracing demonstrated that EA strengthened synaptic integration of the transplanted NN with the host neural circuit. The combination therapy also promoted axonal regeneration, spinal conductivity, and functional recovery. The findings highlight EA as a potential and safe supplementary therapeutic strategy to reinforce the survival and synaptogenesis of a transplanted NN as a neuronal relay to bridge the two severed ends of an injured spinal cord.

A Modular Assembly of Spinal Cord-Like Tissue Allows Targeted Tissue Repair in the Transected Spinal Cord.

Tissue engineering-based neural construction holds promise in providing organoids with defined differentiation and therapeutic potentials. Here, a bioengineered transplantable spinal cord-like tissue (SCLT) is assembled in vitro by simulating the white matter and gray matter composition of the spinal cord using neural stem cell-based tissue engineering technique. Whether the organoid would execute targeted repair in injured spinal cord is evaluated. The integrated SCLT, assembled by white matter-like tissue (WMLT) module and gray matter-like tissue (GMLT) module, shares architectural, phenotypic, and functional similarities to the adult rat spinal cord. Organotypic coculturing with the dorsal root ganglion or muscle cells shows that the SCLT embraces spinal cord organogenesis potentials to establish connections with the targets, respectively. Transplantation of the SCLT into the transected spinal cord results in a significant motor function recovery of the paralyzed hind limbs in rats. Additionally, targeted spinal cord tissue repair is achieved by the modular design of SCLT, as evidenced by an increased remyelination in the WMLT area and an enlarged innervation in the GMLT area. More importantly, the pro-regeneration milieu facilitates the formation of a neuronal relay by the donor neurons, allowing the conduction of descending and ascending neural inputs.

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.

Neurotrophin-3 released from implant of tissue-engineered fibroin scaffolds inhibits inflammation, enhances nerve fiber regeneration, and improves motor function in canine spinal cord injury.

Spinal cord injury (SCI) normally results in cell death, scarring, cavitation, inhibitory molecules release, etc., which are regarded as a huge obstacle to reconnect the injured neuronal circuits because of the lack of effective stimulus. In this study, a functional gelatin sponge scaffold was used to inhibit local inflammation, enhance nerve fiber regeneration, and improve neural conduction in the canine. This scaffold had good porosity and modified with neurotrophin-3 (NT-3)/fibroin complex, which showed sustained release in vitro. After the scaffold was transplanted into canine spinal cord hemisection model, hindlimb movement, and neural conduction were improved evidently. Migrating host cells, newly formed neurons with associated synaptic structures together with functional blood vessels with intact endothelium in the regenerating tissue were identified. Taken together, the results demonstrated that using bioactive scaffold could establish effective microenvironment stimuli for endogenous regeneration, providing a potential and practical strategy for treatment of spinal cord injury. © 2018 The Authors Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2158-2170, 2018.

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.

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.

Cell Transplantation and Neuroengineering Approach for Spinal Cord Injury Treatment: A Summary of Current Laboratory Findings and Review of Literature.

Spinal cord injury (SCI) can cause severe traumatic injury to the central nervous system (CNS). Current therapeutic effects achieved for SCI in clinical medicine show that there is still a long way to go to reach the desired goal of full or significant functional recovery. In basic medical research, however, cell transplantation, gene therapy, application of cytokines, and biomaterial scaffolds have been widely used and investigated as treatments for SCI. All of these strategies when used separately would help rebuild, to some extent, the neural circuits in the lesion area of the spinal cord. In light of this, it is generally accepted that a combined treatment may be a more effective strategy. This review focuses primarily on our recent series of work on transplantation of Schwann cells and adult stem cells, and transplantation of stem cell-derived neural network scaffolds with functional synapses. Arising from this, an artificial neural network (an exogenous neuronal relay) has been designed and fabricated by us-a biomaterial scaffold implanted with Schwann cells modified by the neurotrophin-3 (NT-3) gene and adult stem cells modified with the TrkC (receptor of NT-3) gene. More importantly, experimental evidence suggests that the novel artificial network can integrate with the host tissue and serve as an exogenous neuronal relay for signal transfer and functional improvement of SCI.

Graft of the NT-3 persistent delivery gelatin sponge scaffold promotes axon regeneration, attenuates inflammation, and induces cell migration in rat and canine with spinal cord injury.

Persistent neurotrophic factor delivery is crucial to create a microenvironment for cell survival and nerve regeneration in spinal cord injury (SCI). This study aimed to develop a NT-3/fibroin coated gelatin sponge scaffold (NF-GS) as a novel controlled artificial release therapy for SCI. In vitro, bone marrow-derived mesenchymal stem cells (MSCs) were planted into the NF-GS and release test showed that NF-GS was capable to generate a sustainable NT-3 release up to 28 days. MSCs in NF-GS had high cell activity with excellent cell distribution and phenotype. Then, the NF-GS was transplanted into the injury site of spinal cord of rat and canine in vivo, which exhibited strong biocompatibility during post-transplantation period. Four weeks following transplantation, the concentration of NT-3 was much higher than that in control groups. Cavity areas in the injury/graft site were significantly reduced due to tissue regeneration and axonal extensions associated with myelin sheath through the glial scar into the NF-GS. Additionally, the NF-GS decreased the inflammation by reducing the CD68 positive cells and TNF-α. A striking feature was the occurrence of some cells and myelin-like structure that appeared to traverse the NF-GS. The present results demonstrate that the NF-GS has the property to control the release of NT-3 from the NT-3/fibroin complex thus facilitating regeneration of injured spinal cord.