William A. Pennant

Hypoxia-preconditioned adipose tissue-derived mesenchymal stem cell increase the survival and gene expression of engineered neural stem cells in a spinal cord injury model.

Hypoxic preconditioning (HP) is a novel strategy to make stem cells resistant to the ischemic environment they encounter after transplantation into injured tissue; this strategy improves survival of both the transplanted cells and the host cells at the injury site. Using both in vitro and in vivo injury models, we confirmed that HP-treated adipose tissue-derived mesenchymal stem cells (HP-AT-MSCs) increased cell survival and enhanced the expression of marker genes in DsRed-engineered neural stem cells (NSCs-DsRed). Similar to untreated AT-MSCs, HP-AT-MSCs had normal morphology and were positive for the cell surface markers CD90, CD105, and CD29, but not CD31. In three in vitro ischemic-mimicking injury models, HP-AT-MSCs significantly increased both the viability of NSCs-DsRed and the expression of DsRed and clearly reduced the number of annexin-V-positive apoptotic NSCs-DsRed and the expression of the apoptotic factor Bax. Consistent with the in vitro assay, co-transplantation of NSCs-DsRed with HP-AT-MSCs significantly improved the survival of the NSCs-DsRed, resulting in an increased expression of the DsRed reporter gene at the transplantation site in a rat spinal cord injury (SCI) model. These findings suggest that the co-transplantation of HP-AT-MSCs with engineered NSCs can improve both the cell survival and the gene expression of the engineered NSCs, indicating that this novel strategy can be used to augment the therapeutic efficacy of combined stem cell and gene therapies for SCI.

Cotransplantation of mouse neural stem cells (mNSCs) with adipose tissue-derived mesenchymal stem cells improves mNSC survival in a rat spinal cord injury model.

The low survival rate of graft stem cells after transplantation into recipient tissue is a major obstacle for successful stem cell therapy. After transplantation into the site of spinal cord injury, the stem cells face not only hypoxia due to low oxygen conditions, but also a lack of nutrients caused by damaged tissues and poor vascular supply. To improve the survival of therapeutic stem cells after grafting into the injured spinal cord, we examined the effects of cotransplanting mouse neural stem cells (mNSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs) on mNSC viability. The viability of mNSCs in coculture with AT-MSCs was significantly increased compared to mNSCs alone in an in vitro injury model using serum deprivation (SD), hydrogen peroxide (H2O2), and combined (SD + H2O2) injury mimicking the ischemic environment of the injured spinal cord. We demonstrated that AT-MSCs inhibited the apoptosis of mNSCs in SD, H2O2, and combined injury models. Consistent with these in vitro results, mNSCs transplanted into rat spinal cords with AT-MSCs showed better survival rates than mNSCs transplanted alone. These findings suggest that cotransplantation of mNSCs with AT-MSCs may be a more effective transplantation protocol to improve the survival of cells transplanted into the injured spinal cord.

Robot-assisted Resection of Paraspinal Schwannoma

Resection of retroperitoneal tumors is usually perfomed using the anterior retroperitoneal approach. Our report presents an innovative method utilizing a robotic surgical system. A 50-yr-old male patient visited our hospital due to a known paravertebral mass. Magnetic resonance imaging showed a well-encapsulated mass slightly abutting the abdominal aorta and left psoas muscle at the L4-L5 level. The tumor seemed to be originated from the prevertebral sympathetic plexus or lumbosacral trunk and contained traversing vessels around the tumor capsule. A full-time robotic transperitoneal tumor resection was performed. Three trocars were used for the robotic camera and working arms. The da Vinci Surgical System® provided delicate dissection in the small space and the tumor was completely removed without damage to the surrounding organs and great vessels. This case demonstrates the feasibility of robotic resection in retroperitoneal space. Robotic surgery offered less invasiveness in contrast to conventional open surgery.

Robot-Assisted Transoral Odontoidectomy : Experiment in New Minimally Invasive Technology, a Cadaveric Study

OBJECTIVE In the field of spinal surgery, a few laboratory results or clinical cases about robotic spinal surgery have been reported. In vivo trials and development of related surgical instruments for spinal surgery are required before its clinical application. We investigated the use of the da Vinci® Surgical System in spinal surgery at the craniovertebral junction in a human cadaver to demonstrate the efficacy and pitfalls of robotic surgery. METHODS Dissection of pharyngeal wall to the exposure of C1 and odontoid process was performed with full robotic procedure. Although assistance of another surgeon was necessary for drilling and removal of odontoid process due to the lack of appropriate end-effectors, successful robotic procedures for dural sutures and exposing spinal cord proved its safety and dexterity. RESULTS Robot-assisted odontoidectomy was successfully performed in a human cadaver using the da Vinci® Surgical System with few robotic arm collisions and minimal soft tissue damages. Da Vinci® Surgical System manifested more dexterous movement than human hands in the deep and narrow oral cavity. Furthermore, sutures with robotic procedure in the oral cavity demonstrated the advantage over conventional procedure. CONCLUSION Presenting cadaveric study proved the probability of robot-assisted transoral approach. However, the development of robotic instruments specific to spinal surgery must first precede its clinical application.

Controlled nonviral gene delivery and expression using stable neural stem cell line transfected with a hypoxia-inducible gene expression system.

Nonviral ex vivo local gene therapy systems consisting of regulated gene expression vectors and cellular delivery platforms represent a novel strategy for tissue repair and regeneration. We introduced a hypoxia‐regulated plasmid‐based system into mouse neural stem cells (NSCs) as an efficient gene expression and delivery platform for rapid, robust and persistent hypoxic/ischemic‐regulated gene expression in the spinal cord.

Neural stem cells modified by a hypoxia-inducible VEGF gene expression system improve cell viability under hypoxic conditions and spinal cord injury.

Study Design. An in vitro neural hypoxia model and rat spinal cord injury (SCI) model were used to assess the regulation of therapeutic vascular endothelial growth factor (VEGF) gene expression in mouse neural stem cells (mNSCs) by the EPO (erythropoietin) enhancer or RTP801 promoter. Objective. To increase VEGF gene expression in mNSCs under hypoxic conditions in SCI lesions but avoid unwanted overexpression of VEGF in normal sites, we developed a hypoxia-inducible gene expression system consisting of the EPO enhancer and RTP801 promoter fused to VEGF or the luciferase gene, then transfected into mNSCs. Summary of Background Data. On the basis of the ischemic response in the injured area, poor cell survival at the transplantation site is a consistent problem with NSC transplantation after SCI. Although VEGF directly protects neurons and enhances neurite outgrowth, uncontrolled overexpression of VEGF in uninjured tissue may cause serious adverse effects. To effectively improve NSC survival in ischemic sites after transplantation, we evaluated mNSCs modified by a hypoxia-inducible VEGF gene expression system in an SCI model. Methods. Hypoxia-inducible luciferase or VEGF plasmids were constructed using the EPO enhancer or RTP801 promoter. The effect of these systems on targeted gene expression and cell viability was evaluated in mNSCs in both hypoxic in vitro injury and a rat SCI model in vivo. Results. The gene expression system containing the EPO enhancer or RTP801 promoter significantly increased the expression of the luciferase reporter gene and therapeutic VEGF gene under hypoxic conditions. The Epo-SV-VEGF plasmid transfection group had significantly fewer apoptotic cells in vitro. This system also augmented cell viability in the in vivo SCI model. Conclusion. These results strongly suggest the potential utility of mNSCs modified by a hypoxia-inducible VEGF gene expression system in the development of effective stem cell transplantation protocols in SCI.

Chitosan/TPP-Hyaluronic Acid Nanoparticles: A New Vehicle for Gene Delivery to the Spinal Cord

Abstract Gene delivery offers therapeutic promise for the treatment of neurological diseases and spinal cord injury. Several studies have offered viral vectors as vehicles to deliver therapeutic agents, yet their toxicity and immunogenicity, along with the cost of their large-scale formulation, limits their clinical use. As such, non-viral vectors are attractive in that they offer improved safety profiles compared to viruses. Poly(ethylene imine) (PEI) is one of the most extensively studied non-viral vectors, but its clinical value is limited y its cytotoxicity. Recently, chitosan/DNA complex nanoparticles have een considered as a vector for gene delivery. Here, we demonstrate that DNA nanoparticles made of hyaluronic acid (HA) and chitosan have low cytotoxicity and induce high transgene expression in neural stem cells and organotypic spinal cord slice tissue. Chitosan-TPP/HA nanoparticles were significantly less cytotoxic than PEI at various concentrations. Additionally, chitosan-TPP/HA nanoparticles with pDNA induced higher transgene expression in vitro for a longer duration than PEI in neural stem cells. These results suggest chitosan-TPP/HA nanoparticles may have the potential to serve as an option for gene delivery to the spinal cord.

Co-transplantation of bone marrow-derived mesenchymal stem cells and nanospheres containing FGF-2 improve cell survival and neurological function in the injured rat spinal cord

BackgroundSpinal cord injury (SCI) is a devastating and irreversible event, and much research using fibroblast growth factor-2 (FGF-2) has been performed to test its capacity to blunt the effects of SCI as well as to provide an environment conducive for SCI repair.MethodsWe tested how the in vitro release of FGF-2 from heparin-conjugated poly(L-lactide-co-glycolide) (PLGA)-conjugated nanospheres (HCPNs) affected the growth of human bone marrow-derived mesenchymal stem cells (hBMSCs), as well as the effects of their co-transplantation in an animal model of SCI.ResultsOur results showed that sustained, long-term release of FGF-2 from HCPNs significantly increased hBMSCs proliferation in vitro, and that their co-transplantation following rat SCI lead to increased functional improvement, a greater amount of hBMSCs surviving transplantation, and a greater density of neurofilament-positive cells in the injury epicenter.ConclusionThese results suggest a proliferative, protective, and neural inductive potential of FGF-2 for transplanted hBMSCs, as well as a possible role for sustained FGF-2 delivery along with hBMSCs transplantation in the injured spinal cord. Future studies will be required to ascertain the safety FGF-2-containing HCPNs before clinical application.

Hypoxia-specific VEGF-expressing Neural Stem Cells in Spinal Cord Injury Model

We established three stable neural stem cell (NSC) lines to explore the possibility of using hypoxia-specific vascular endothelial growth factor (VEGF) expressing NSC lines (EpoSV-VEGF NSCs) to treat spinal cord injury. The application of EpoSV-VEGF NSCs into the injured spinal cord after clip compression injury not only showed therapeutic effects such as extended survival and angiogenesis, but also displayed its safety profile as it did not cause unwanted cell proliferation or angiogenesis in normal spinal cord tissue, as EpoSV-VEGF NSCs consistently showed hypoxia-specific VEGF expression patterns. This suggests that our EpoSV-VEGF NSCs are both safe and therapeutically efficacious for the treatment of spinal cord injury. Furthermore, this hypoxia-inducible gene expression system may represent a safe tool suitable for gene therapy.

Hypoxia-induced expression of VEGF in the organotypic spinal cord slice culture.

We used the erythropoietin enhancer and Simian virus-40 promoter to create a hypoxia-inducible gene expression system to investigate the effect of vascular endothelial growth factor (VEGF) gene therapy on neuroprotection and neurogenesis in organotypic spinal cord slice culture. The organotypic spinal cord slice culture transfected with pEpo-SV-VEGF expressed the highest amount of VEGF under hypoxic conditions and showed decreased apoptosis and increased proliferation, and evidence of neurogenesis. Our results show that the hypoxia-induced VEGF expression in an organotypic spinal cord slice culture may lead to an optimal treatment for spinal cord injury.