Co-Administration of Resolvin D1 and Peripheral Nerve-Derived Stem Cell Spheroids as a Therapeutic Strategy in a Rat Model of Spinal Cord Injury.

Spinal cord injury (SCI), primarily caused by trauma, leads to permanent and lasting loss of motor, sensory, and autonomic functions. Current therapeutic strategies are focused on mitigating secondary injury, a crucial aspect of SCI pathophysiology. Among these strategies, stem cell therapy has shown considerable therapeutic potential. This study builds on our previous work, which demonstrated the functional recovery and neuronal regeneration capabilities of peripheral nerve-derived stem cell (PNSC) spheroids, which are akin to neural crest stem cells, in SCI models. However, the limited anti-inflammatory capacity of PNSC spheroids necessitates a combined therapeutic approach. As a result, we investigated the potential of co-administering resolvin D1 (RvD1), known for its anti-inflammatory and neuroprotective properties, with PNSC spheroids. In vitro analysis confirmed RvD1's anti-inflammatory activity and its inhibitory effect on pro-inflammatory cytokines. In vivo studies involving a rat SCI model demonstrated that combined therapy of RvD1 and PNSC spheroids outperformed monotherapies, exhibiting enhanced neuronal regeneration and anti-inflammatory effects as validated through behavior tests, quantitative reverse transcription polymerase chain reaction, and immunohistochemistry. Thus, our findings suggest that the combined application of RvD1 and PNSC spheroids may represent a novel therapeutic approach for SCI management.

Heparin-Based Hydrogel Micropatches with Human Adipose-Derived Stem Cells: A Promising Therapeutic Approach for Neuropathic Pain Relief.

This study explores the therapeutic efficacy of heparin-based hydrogel micropatches containing human adipose-derived stem cells (hASCs) in treating neuropathic pain caused by nerve damage. Our results showed that hASCs exhibited neuroregenerative and pain-relieving effects when used with heparin-based hydrogel micropatches in the neuropathic pain animal model. The use of this combination also produced enhanced cell viability and nerve regeneration. We conducted various neurological behavioral tests, dynamic plantar tests, histological examinations, and neuroelectrophysiological examinations to confirm the therapeutic effect. Our findings suggest that this approach could maximize therapeutic efficacy and improve the quality of life for patients suffering from neuropathic pain.

Multi-modulation of immune-inflammatory response using bioactive molecule-integrated PLGA composite for spinal fusion.

Despite current developments in bone substitute technology for spinal fusion, there is a lack of adequate materials for bone regeneration in clinical applications. Recombinant human bone morphogenetic protein-2 (rhBMP-2) is commercially available, but a severe inflammatory response is a known side effect. Bone graft substitutes that enhance osteogenesis without adverse effects are needed. We developed a bioactive molecule-laden PLGA composite with multi-modulation for bone fusion. This bioresorbable composite scaffold was considered for bone tissue engineering. Among the main components, magnesium hydroxide (MH) aids in reduction of acute inflammation affecting disruption of new bone formation. Decellularized bone extracellular matrix (bECM) and demineralized bone matrix (DBM) composites were used for osteoconductive and osteoinductive activities. A bioactive molecule, polydeoxyribonucleotide (PDRN, PN), derived from trout was used for angiogenesis during bone regeneration. A nano-emulsion method that included Span 80 was used to fabricate bioactive PLGA-MH-bECM/DBM-PDRN (PME2/PN) composite to obtain a highly effective and safe scaffold. The synergistic effect provided by PME2/PN improved not only osteogenic and angiogenic gene expression for bone fusion but also improved immunosuppression and polarization of macrophages that were important for bone tissue repair, using a rat model of posterolateral spinal fusion (PLF). It thus had sufficient biocompatibility and bioactivity for spinal fusion.

Concentration-Dependent Efficacy of Recombinant Human Bone Morphogenetic Protein-2 Using a HA/ß-TCP Hydrogel Carrier in a Mini-Pig Vertebral Oblique Lateral Interbody Fusion Model.

Bone morphogenetic protein-2 (BMP-2) is used in the treatment of degenerative spinal disease and vertebral fractures, spine fusion, dental surgery, and facial surgery. However, high doses are associated with side effects such as inflammation and osteophytes. In this study, we performed spinal fusion surgery on mini-pigs using BMP-2 and a HA/ß-TCP hydrogel carrier, and evaluated the degree of fusion and osteophyte growth according to time and dosage. Increasing the dose of BMP-2 led to a significantly higher fusion rate than was observed in the control group, and there was no significant difference between the 8-week and 16-week samples. We also found that the HA + ß-TCP hydrogel combination helped maintain the rate of BMP-2 release. In conclusion, the BMP-2-loaded HA/ß-TCP hydrogel carrier used in this study overcame the drawback of potentially causing side effects when used at high concentrations by enabling the sustained release of BMP-2. This method is also highly efficient, since it provides mineral matter to accelerate the fusion rate of the spine and improve bone quality.

Peripheral Nerve-Derived Stem Cell Spheroids Induce Functional Recovery and Repair after Spinal Cord Injury in Rodents.

Stem cell therapy is one of the most promising candidate treatments for spinal cord injury. Research has shown optimistic results for this therapy, but clinical limitations remain, including poor viability, engraftment, and differentiation. Here, we isolated novel peripheral nerve-derived stem cells (PNSCs) from adult peripheral nerves with similar characteristics to neural-crest stem cells. These PNSCs expressed neural-crest specific markers and showed multilineage differentiation potential into Schwann cells, neuroglia, neurons, and mesodermal cells. In addition, PNSCs showed therapeutic potential by releasing the neurotrophic factors, including glial cell-line-derived neurotrophic factor, insulin-like growth factor, nerve growth factor, and neurotrophin-3. PNSC abilities were also enhanced by their development into spheroids which secreted neurotrophic factors several times more than non-spheroid PNSCs and expressed several types of extra cellular matrix. These features suggest that the potential for these PNSC spheroids can overcome their limitations. In an animal spinal cord injury (SCI) model, these PNSC spheroids induced functional recovery and neuronal regeneration. These PNSC spheroids also reduced the neuropathic pain which accompanies SCI after remyelination. These PNSC spheroids may represent a new therapeutic approach for patients suffering from SCI.

Hypoxia-specific, VEGF-expressing neural stem cell therapy for safe and effective treatment of neuropathic pain.

Vascular endothelial growth factor (VEGF) is an angiogenic cytokine that stimulates the differentiation and function of vascular endothelial cells. VEGF has been implicated in improving nervous system function after injury. However, uncontrolled overexpression of VEGF increases the risk of tumor formation at the site of gene delivery. For this reason, VEGF expression needs to be strictly controlled. The goal of the present study was to understand the effects of hypoxia-induced gene expression system to control VEGF gene expression in neural stem cells (NSCs) on the regeneration of neural tissue after sciatic nerve injury. In this study, we used the erythropoietin (Epo) enhancer-SV40 promoter system (EpoSV-VEGF-NSCs) for hypoxia-specific VEGF expression. We used three types of NSCs: DsRed-NSCs as controls, SV-VEGF-NSCs as uncontrolled VEGF overexpressing NSCs, and EpoSV-VEGF-NSCs. For comparison of VEGF expression at normoxia and hypoxia, we measured the amount of VEGF secreted. VEGF expression decreased at normoxia and increased at hypoxia for EpoSV-VEGF-NSCs; thus, EpoSV-VEGF-NSCs controlled VEGF expression, dependent upon oxygenation condition. To demonstrate the therapeutic effect of EpoSV-VEGF-NSCs, we transplanted each cell line in a neuropathic pain sciatic nerve injury rat model. The transplanted EpoSV-VEGF-NSCs improved sciatic nerve functional index (SFI), mechanical allodynia, and re-myelination similar to the SV-VEGF-NSCs. Additionally, the number of blood vessels increased to a level similar to that of the SV-VEGF-NSCs. However, we did not observe tumor generation in the EpoSV-VEGF-NSC animals that were unlikely to have tumor formation in the SV-VEGF-NSCs. From our results, we determined that EpoSV-VEGF-NSCs safely regulate VEGF gene expression which is dependent upon oxygenation status. In addition, we found that they are therapeutically appropriate for treating sciatic nerve injury.

Antiapoptotic Effect of Highly Secreted GMCSF From Neuronal Cell-specific GMCSF Overexpressing Neural Stem Cells in Spinal Cord Injury Model.

STUDY DESIGN: Neuronal cell-specific gene expression system and neural stem cells (NSCs) were combined for treatment of spinal cord injury (SCI). OBJECTIVE: To verify the reproducibility of the neuronal cell-specific therapeutic gene overexpression system, we develop a neuronal cell-specific granulocyte-macrophage colony-stimulating factor expression system (NSE-GMCSF), and then examine the characteristics of GMCSF overexpression and protective effect on neural cells in vitro and vivo. SUMMARY OF BACKGROUND DATA: The stem cell transplantation is considered a promising therapy for SCI. However, stem cell monotherapy strategy is insufficient for complete recovery after SCI. Therefore, combined treatment method based on stem cells with other therapeutic system may be effective for improving the therapeutic efficacy. In this study, we established the gene and stem cell therapy platform based on NSCs and neuronal cell-specific gene expression system. METHODS: To examine the GMCSF expression pattern, we compared the amount of secreted GMCSF from the neuronal cell-specific GMCSF expressing NSCs with control GMCSF-expressing NSCs (respectively, NSE-GMCSF-NSCs vs. SV-GMCSF-NSCs) by ELISA in vitro and in vivo, and then verified the neuronal protective effect of these cells in vitro and vivo. RESULTS: The results showed that NSE-GMCSF-NSCs secreted more GMCSF compared with SV-GMCSF-NSCs in normoxia, hypoxia and cytotoxic conditions. The cell viability of NSE-GMCSF-NSCs was increased depending on the amount of secreted GMCSF in cytotoxic condition. In addition, the amount of secreted GMCSF by NSE-GMCSF-NSCs transplanted into injured spinal cord was significantly higher than SV-GMCSF-NSCs. Higher amount of secreted GMCSF decreased the expression of proapoptotic protein, Bax. CONCLUSION: In this study, we demonstrated that the neuronal cell-specific gene expression system induced overexpression of GMCSF in NSCs. These combined NSCs & gene therapy treatment protocol would be an effective therapeutic system for SCI. LEVEL OF EVIDENCE: N/A.

A Gene and Neural Stem Cell Therapy Platform Based on Neuronal Cell Type-Inducible Gene Overexpression.

PURPOSE: Spinal cord injury (SCI) is associated with permanent neurological damage, and treatment thereof with a single modality often does not provide sufficient therapeutic outcomes. Therefore, a strategy that combines two or more techniques might show better therapeutic effects. MATERIALS AND METHODS: In this study, we designed a combined treatment strategy based on neural stem cells (NSCs) introduced via a neuronal cell type-inducible transgene expression system (NSE::) controlled by a neuron-specific enolase (NSE) promoter to maximize therapeutic efficiency and neuronal differentiation. The luciferase gene was chosen to confirm whether this combined system was working properly prior to using a therapeutic gene. The luciferase expression levels of NSCs introduced via the neuronal cell type-inducible luciferase expression system (NSE::Luci) or via a general luciferase expressing system (SV::Luci) were measured and compared in vitro and in vivo. RESULTS: NSCs introduced via the neuronal cell type-inducible luciferase expressing system (NSE::Luci-NSCs) showed a high level of luciferase expression, compared to NSCs introduced via a general luciferase expressing system (SV::Luci-NSCs). Interestingly, the luciferase expression level of NSE::Luci-NSCs increased greatly after differentiation into neurons. CONCLUSION: We demonstrated that a neuronal cell type-inducible gene expression system is suitable for introducing NSCs in combined treatment strategies. We suggest that the proposed strategy may be a promising tool for the treatment of neurodegenerative disorders, including SCI.

Human Adipose Stem Cells Improve Mechanical Allodynia and Enhance Functional Recovery in a Rat Model of Neuropathic Pain.

Stem cells are a promising source of tissue engineering due to their differentiation potential. Today, direct transplantation of stem cells for cell therapy is commonly performed. However, in cases of nerve injury, direct transplantation of cells could lead to secondary nerve damage. Male Sprague-Dawley rats were randomized into four groups: the phosphate-buffered saline epineural transplantation (PBS-ENT) group, the PBS intraneural transplantation (PBS-INT) group, the human adipose-derived stem cells epineural transplantation (hASCs-ENT) group, and human adipose-derived stem cells intraneural transplantation (hASCs-INT) group. Transplantation was conducted 1 week later after inflicting a crush injury with subsequent observation for 5 weeks. To evaluate pain, each group was examined with regard to paw withdrawal latency and evoked potentials. The sciatic functional index (SFI) was calculated to estimate functional recovery. The sciatic nerve was also examined histologically. The hASCs-ENT group showed a more rapid paw withdrawal threshold and SFI recovery than the other groups (p<0.05). The hASCs-ENT group also showed shorter initial latencies in both somatosensory evoked potential (SSEP) and motor evoked potential (MEP) than the PBS-INT group (p<0.05). In addition, the N1 latency of the MEP and the N1 and P1 latencies of the SSEP were significantly shorter than those of the PBS-INT group (p<0.05). Histological examination revealed that the transplanted groups showed better neural recovery and remyelination than the groups injected with PBS. These results show that the transplantation of hASCs into the injured sciatic nerve improved mechanical allodynia and functional recovery as determined by the paw withdrawal test, SFI analysis, and electrophysiological studies. ENT is superior to INT in terms of invasiveness and better outcomes.

Vascular endothelial growth factor-expressing neural stem cell for the treatment of neuropathic pain.

Previously, we determined that vascular endothelial growth factor (VEGF) improves the survival of neural stem cells (NSCs) transplanted into an ischemic environment and effectively enhances angiogenesis. Here, we applied NSCs expressing VEGF (SV-VEGF-NSCs) to treat neuropathic pain. In this study, our goal was to verify the therapeutic effect of SV-VEGF-NSCs by transplanting the cells in a sciatic nerve injury model. We compared the amount of VEGF secreted from DsRed-NSCs (control) or SV-VEGF-NSCs and observed that SV-VEGF-NSCs have a much higher expression level of VEGF. We next investigated whether transplantation with SV-VEGF-NSCs aids functional recovery and pain reduction. We confirmed that transplantation with SV-VEGF-NSCs enhances functional recovery, pain reduction, and remyelination as well as the number of blood vessels compared with the control groups. Our results show that VEGF aids functional recovery and pain reduction in a sciatic nerve injury model.

Enhancement of mitochondrial function correlates with the extension of lifespan by caloric restriction and caloric restriction mimetics in yeast.

Caloric restriction mimetics (CRMs) have been developed to mimic the effects of caloric restriction (CR). However, research reports for the effects of CRMs are often times inconsistent across different research groups. Therefore, in this study, we compared seven identified CRMs which extend the lifespans of various organisms including caffeine, curcumin, dapsone, metformin, rapamycin, resveratrol, and spermidine to CR for mitochondrial function in a single model, Saccharomyces cerevisiae. In this organism, rapamycin extended chronological lifespan (CLS), but other CRMs failed to extend CLS. Rapamycin enhanced mitochondrial function like CR did, but other CRMs did not. Both CR and rapamycin worked on mitochondrial function, but they worked at different windows of time during the chronological aging process.