BST2, a Novel Inhibitory Receptor, Is Involved in NK Cell Cytotoxicity through Its Cytoplasmic Tail Domain.

Bone Marrow Stromal Cell Antigen 2 (BST2) is a type II transmembrane protein expressed on various cell types that tethers the release of viruses. Natural killer (NK) cells express low levels of BST2 under normal conditions but exhibit increased expression of BST2 upon activation. In this study, we show for the first time that murine BST2 can control the cytotoxicity of NK cells. The cytoplasmic tail of murine BST2 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM). The absence of BST2 on NK cells can enhance their cytotoxicity against tumor cells compared to wild type NK cells. NK cells isolated from NZW mice, which express ITIM-deficient BST2, also showed higher cytotoxicity than wild type NK cells. In addition, we found that galectin-8 and galectin-9 were ligands of BST2, since blocking galectin-8 or -9 with monoclonal antibodies enhanced the cytotoxicity of NK cells. These results suggested that BST2 might be a novel NK cell inhibitory receptor as it was involved in regulating NK cell cytotoxicity through its interaction with galectins.

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.

BST2 inhibits infection of influenza A virus by promoting apoptosis of infected cells.

BST2 is an antiviral factor that inhibits the release of enveloped virus at the plasma membrane via an unusual topology in which its N-terminal is in the cytosol while its C-terminal is anchored by glycophosphatidylinositol (GPI). BST2-deficient cells showed substantially higher release of virions than wild type cells. Influenza-infected BST2-deficient cells showed greatly reduced cytopathic effect (CPE) than wild type cells despite their generally robust virus production. This finding prompted us to determine whether BST2 was involved in the apoptotic process of virus-infected host cells. Our results revealed that BST2 might be involved in IRE1α-mediated ER stress pathway by increasing spliced form XBP-1. Consequently, levels of cytochrome C, caspase-3, caspase-9, and PARP as representative molecules of apoptosis were significantly increased in wild type cells than those in BST2-deficient cells. These results suggest that BST2 might participate in innate host defense by augmenting ER-stress-induced apoptotic signaling to inhibit the replication and spread of virus.

Enhanced production of enveloped viruses in BST-2-deficient cell lines.

Despite all the advantages that cell-cultured influenza vaccines have over egg-based influenza vaccines, the inferior productivity of cell-culture systems is a major drawback that must be addressed. BST-2 (tetherin) is a host restriction factor which inhibits budding-out of various enveloped viruses from infected host cells. We developed BST-2-deficient MDCK and Vero cell lines to increase influenza virus release in cell culture. BST-2 gene knock-out resulted in increased release of viral particles into the culture medium, by at least 2-fold and up to 50-fold compared to release from wild-type counterpart cells depending on cell line and virus type. The effect was not influenza virus/MDCK/Vero-specific, but was also present in a broad range of host cells and virus families; we observed similar results in murine, human, canine, and monkey cell lines with viruses including MHV-68 (Herpesviridae), influenza A virus (Orthomyxoviridae), porcine epidemic diarrhea virus (Coronaviridae), and vaccinia virus (Poxviridae). Our results suggest that the elimination of BST-2 expression in virus-producing cell lines can enhance the production of viral vaccines. Biotechnol. Bioeng.2017;114: 2289-2297. © 2017 Wiley Periodicals, Inc.

LncRNA TSIX knockdown restores spinal cord injury repair through miR-30a/SOCS3 axis.

Spinal cord injury (SCI) is a serious injury to the central nervous system. Previous studies have discovered that the development of SCI is associated with gene expression. The purpose of this study was to explore the significance of lncRNA TSIX in SCI and its underlying mechanism involved. An in vivo SCI mice model and an in vitro hypoxia-treated HT22 cells model were applied in this study. TSIX and SOCS3 expression in SCI tissues was measured by qRT-PCR, western blot and FISH assay. LV-sh-TSIX was injected into SCI mice intrathecally or subjected to HT22 cells to access the consequent alteration in inflammation response, cell apoptosis and functional recovery through ELISA, immunohistochemistry, TUNEL, flow cytometry assays and BMS scores. Then, the underlying mechanism of TSIX was analyzed by bioinformatics analysis and then confirmed by RIP, RNA pull-down and dual-luciferase reporter assay. It was identified that TSIX was up-regulated in HT22 cells under hypoxia operation and spinal cord tissues of SCI mice. TSIX knockdown improved the lesion size and BMS score and inhibited inflammation and cell apoptosis. MiR-30a was identified as a target for TSIX and SOCS3, and TSIX binds to miR-30a by competing with SOCS3, thereby counteracting miR-30a-mediated SOCS3 inhibition. In addition, LV-sh-TSIX effects were significantly overturned by miR-30a inhibition or SOCS3 over-expression. Knockdown of TSIX improved functional recovery and attenuated the inflammation response and cell apoptosis via miR-30a/SOCS3 axis. These results may provide a potential novel insight for SCI treatment.

Combined Method of Neuronal Cell-Inducible Vector and Valproic Acid for Enhanced Gene Expression under Hypoxic Conditions.

BACKGROUND: Gene therapy shows the ability to restore neuronal dysfunction via therapeutic gene expression. The efficiency of gene expression and delivery to hypoxic injury sites is important for successful gene therapy. Therefore, we established a gene/stem cell therapy system using neuron-specific enolase promoter and induced neural stem cells in combination with valproic acid to increase therapeutic gene expression in hypoxic spinal cord injury. METHODS: To examine the effect of combined method on enhancing gene expression, we compared neuronal cell-inducible luciferase levels under normoxia or hypoxia conditions in induced neural stem cells with valproic acid. Therapeutic gene, vascular endothelial growth factor, expression with combined method was investigated in hypoxic spinal cord injury model. We verified gene expression levels and the effect of different methods of valproic acid administration in vivo. RESULTS: The results showed that neuron-specific enolase promoter enhanced gene expression levels in induced neural stem cells compared to Simian Virus 40 promoter under hypoxic conditions. Valproic acid treatment showed higher gene expression of neuron-specific enolase promoter than without treatment. In addition, gene expression levels and cell viability were different depending on the various concentration of valproic acid. The gene expression levels were increased significantly when valproic acid was directly injected with induced neural stem cells in vivo. CONCLUSION: In this study, we demonstrated that the combination of neuron-specific enolase promoter and valproic acid induced gene overexpression in induced neural stem cells under hypoxic conditions and also in spinal cord injury depending on valproic acid administration in vivo. Combination of valproic acid and neuron-specific enolase promoter in induced neural stem cells could be an effective gene therapy system for hypoxic spinal cord injury.

The combination of forskolin and VPA increases gene expression efficiency to the hypoxia/neuron-specific system.

BACKGROUND: Spinal cord injury (SCI) tends to damage neural tissue and generate a hypoxic environment. Studies have confirmed that single therapy with gene or stem cells is inefficient, but research into combining stem cells and gene therapy in treating tissue damage has been undertaken to overcome the related limitations, which include low gene delivery efficiency and therapeutic outcome. Thus, a combination of stem cells, gene therapy, and a hypoxia-specific system may be useful for the reconstruction of SCI. METHODS: To synergistically treat SCI, a combined platform using a hypoxia/neuron-inducible gene expression system (HNIS) and human induced-neural stem cells (hiNSCs) produced by direct reprogramming was designed. Sox2- or nestin-positive hiNSCs were differentiated to Tuj1-, MAP2-, or NeuN-positive neurons. RESULTS: HNIS showed consistent hypoxia/neuron-specific gene expression in hiNSCs cultured under hypoxia. In particular, the HNIS-hiNSC combined platform revealed a complex pattern with higher gene expression compared with a single platform. In addition, we found that an optimal combination of small molecules, such as CHIR99021, valproic acid (VPA), glycogen synthase kinase-3ß (GSK3ß), and histone deacetylase (HDAC) inhibitors, could significantly enhance gene expression with HNIS-hiNSCs in the hypoxic environment. CONCLUSIONS: This experiment demonstrated that HNIS-hiNSCs combined with GSK3 and HDAC inhibitors may present another promising strategy in the treatment of SCI.

Malignant gliomas can be converted to non­proliferating glial cells by treatment with a combination of small molecules.

Gliomas, the most highly malignant central nervous system tumors, are associated with an extremely poor patient survival rate. Given that gliomas are derived from mutations in glial precursor cells, a considerable number of them strongly react with glial precursor cell­specific markers. Thus, we investigated whether malignant gliomas can be converted to glial cells through the regulation of endogenous gene expression implicated in glial precursor cells. In the present study, we used three small­molecule compounds, [cyclic adenosine monophosphate (cAMP) enhancer, a mammalian target of rapamycin (mTOR) inhibitor, and a bromodomain and extra­terminal motif (BET) inhibitor] for glial reprogramming. Small­molecule­induced gliomas (SMiGs) were not only transformed into exhibiting a glial­specific morphology, but also showed positive reactions with glial­specific markers such as glial fibrillary acidic protein (GFAP), 2',3'­cyclic nucleotide 3'­phosphohydrolase (CNP) and anti­oligodendrocyte (RIP). A microarray analysis indicated that SMiGs exhibited a marked increase in specific gene levels, whereas that of a malignant cancer­specific gene was greatly decreased. Moreover, proliferation of the cells was markedly suppressed after the conversion of malignant glioma cells into glial cells. Our findings confirmed that malignant gliomas can be reprogrammed to non­proliferating glial cells, using a combination of small molecules, and their proliferation can be regulated by their differentiation. We suggest that our small­molecule combination (with forskolin, rapamycin and I­BET151) may be the next generation of anticancer agents that act by reprogramming malignant gliomas to differentiate into glial cells.

Regulation of cAMP and GSK3 signaling pathways contributes to the neuronal conversion of glioma.

Glioma is the most malignant type of primary central nervous system tumors, and has an extremely poor prognosis. One potential therapeutic approach is to induce the terminal differentiation of glioma through the forced expression of pro-neural factors. Our goal is to show the proof of concept of the neuronal conversion of C6 glioma through the combined action of small molecules. We investigated the various changes in gene expression, cell-specific marker expression, signaling pathways, physiological characteristics, and morphology in glioma after combination treatment with two small molecules (CHIR99021, a glycogen synthase kinase 3 [GSK3] inhibitor and forskolin, a cyclic adenosine monophosphate [cAMP] activator). Here, we show that the combined action of CHIR99021 and forskolin converted malignant glioma into fully differentiated neurons with no malignant characteristics; inhibited the proliferation of malignant glioma; and significantly down-regulated gene ontology and gene expression profiles related to cell division, gliogenesis, and angiogenesis in small molecule-induced neurons. In vivo, the combined action of CHIR99021 and forskolin markedly delayed neurological deficits and significantly reduced the tumor volume. We suggest that reprogramming technology may be a potential treatment strategy replacing the therapeutic paradigm of traditional treatment of malignant glioma, and a combination molecule comprising a GSK3 inhibitor and a cAMP inducer could be the next generation of anticancer drugs.

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.

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.

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.

Human-induced pluripotent stem cells generated from intervertebral disc cells improve neurologic functions in spinal cord injury.

INTRODUCTION: Induced pluripotent stem cells (iPSCs) have emerged as a promising cell source for immune-compatible cell therapy. Although a variety of somatic cells have been tried for iPSC generation, it is still of great interest to test new cell types, especially those which are hardly obtainable in a normal situation. METHODS: In this study, we generated iPSCs by using the cells originated from intervertebral disc which were removed during a spinal operation after spinal cord injury. We investigated the pluripotency of disc cell-derived iPSCs (diPSCs) and neural differentiation capability as well as therapeutic effect in spinal cord injury. RESULTS: The diPSCs displayed similar characteristics to human embryonic stem cells and were efficiently differentiated into neural precursor cells (NPCs) with the capability of differentiation into mature neurons in vitro. When the diPSC-derived NPCs were transplanted into mice 9 days after spinal cord injury, we detected a significant amelioration of hindlimb dysfunction during follow-up recovery periods. Histological analysis at 5 weeks after transplantation identified undifferentiated human NPCs (Nestin(+)) as well as early (Tuj1(+)) and mature (MAP2(+)) neurons derived from the transplanted NPCs. Furthermore, NPC transplantation demonstrated a preventive effect on spinal cord degeneration resulting from the secondary injury. CONCLUSION: This study revealed that intervertebral discs removed during surgery for spinal stabilization after spinal cord injury, previously considered a "waste" tissue, may provide a unique opportunity to study iPSCs derived from difficult-to-access somatic cells and a useful therapeutic resource for autologous cell replacement therapy in spinal cord injury.

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.

Rapid recovery of tissue hypoxia by cotransplantation of endothelial cells.

Disruption of blood vessels caused by a spinal cord injury leads to tissue hypoxia. This hypoxic condition reduces the survival of transplanted stem cells, consequentially decreasing the effectiveness of stem cell therapy. In this study, we investigated the correlation between angiogenesis and the survival of transplanted neural stem cells in a spinal cord injury model. Hypoxia-specific luciferase-expressing neural stem cells (EpoSV-Luc NSC) were used as a tool for the detection of hypoxia caused by a spinal cord injury. In vivo, angiogenesis by cotransplantation of endothelial cells quickly recovered tissue hypoxia caused by a spinal cord injury. As a result, cotransplantation of endothelial cells improved the survival of neural stem cells transplanted into the injured spinal cord.

Role of the oxygen-dependent degradation domain in a hypoxia-inducible gene expression system in vascular endothelial growth factor gene therapy.

STUDY DESIGN.: An in vitro neural hypoxia model and rat spinal cord injury (SCI) model were used to assess the regulation effect of a reporter or therapeutic gene expression by an oxygen-dependent degradation (ODD) domain in a hypoxia-inducible gene expression system with or without the erythropoietin (EPO) enhancer. OBJECTIVE.: To increase vascular endothelial growth factor (VEGF) gene expression 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 upstream of the SV promoter and an ODD domain C-terminally fused to VEGF. SUMMARY OF BACKGROUND DATA.: ODD domain plays a major role in the degradation of hypoxia-inducible factor 1alpha and has been used in a hypoxia-specific gene expression system as a post-translational regulatory factor. METHODS.: The hypoxia-inducible luciferase or VEGF plasmid was constructed using the EPO enhancer combined with or without the ODD domain. The constructed plasmid was transfected into mouse Neuro 2a (N2a) neuroblastoma cells by Lipofectamine 2000, followed by a 24-hour incubation in hypoxia or normoxia. For in vivo analysis, the naked plasmid DNA was directly injected into the injured rat spinal cord. The gene expression was evaluated by luciferase activity assay, enzyme-linked immunosorbent assay, reverse transcriptase-polymerase chain reaction, and immunofluorescence staining. RESULTS.: The EPO enhancer/ODD domain-combined hypoxia-inducible gene expression system clearly increased the expression of the reporter luciferase gene and therapeutic VEGF gene specifically under hypoxic conditions and SCI, and quickly downregulated protein expression to a very low level after reoxygenation. CONCLUSION.: These results strongly suggest the potential applicability of this EPO enhancer/ODD domain-based hypoxia-inducible gene expression system in the development of a safer and more effective VEGF gene therapy for SCI.