Glioma Gene Therapy Research Articles

The experimental investigation of glioma-trophic capacity of human umbilical cord-derived mesenchymal stem cells after intraventricular administration

Objective To explore the glioma⁃trophic migration capacity of human umbilical cord-derived mesenchymal stem cells (hUC⁃MSCs) by intraventricular administration. Methods The umbilical cord tissue were obtained during full-term pregnancy cesarean section under sterile conditions. This study was approved by Ethics Committee and got the informed consent of patient. The hUC-MSCs were isolated by trypsin and collagenase digestion, followed by adherent culture methods. The characteristics of isolated hUC-MSCs were demonstrated by cell morphylogy, phenotype analysis and multi-differentiation potentials into adipocytes, osteoblasts and neural cells. Then the hUC-MSCs were labeled with CM-DiI and injected into contralateral ventricle of glioma of the C6 glioma⁃bearing Sprague-Dawley (SD) rats. Two weeks later, the rats were sacrificed and the brains were taken out to examine the migration and distribution of hUC-MSCs in the tumor bed, at the interface of tumor and cerebral parenchyma as well as the tumor satelites infiltrating into the normal brain. Results The hUC-MSCs demonstrated plastic-adherent characterization and homogeneous fibroblastic-like morphylogy in culture, expression of specific surface phenotypes of MSCs (CD13, CD29, CD44, CD90) but not endothelial or hematopoietic markers (CD14, CD31, CD34, CD38, CD45, CD133), and muti-differentiatiation potentials into Oil red O stained adipocytes, Alizarin red S stained osteoblasts, neuron-specific enolase (NSE)-positive neurons and glial fibrillary acidic protein (GFAP)-positive astrocytes in permissive inducive conditions. Importantly, after labeled hUC-MSCs injection into contralateral ventricle of glioma, the hUC-MSCs migrated from initial injection site to the glioma mass and along the interface of tumor and brain, and some of them "chasing" the glioma satellites infiltrated into the normal parenchyma. Conclusion The hUC-MSCs possess prominent tumor-specific targeting capacity and extensive intratumoral distribution in glioma models. Thus, they may serve as novel vehicles in cell-based gene-therapy of glioma.

Poly(amido amine) is an ideal carrier of miR-7 for enhancing gene silencing effects on the EGFR pathway in U251 glioma cells

microRNAs are regarded as promising drugs for glioma gene therapy. However, conventional administration routes, such as oral administration and intravenous infusion, present low efficiency due to the blood-brain barrier and intercellular retention, thereby limiting their application. Recent studies showed poly(amido amine) (PAMAM) was a candidate carrier due to its high solubilization, delayed release and low toxicity. In the present study, U251 human brain glioma cells were transfected with the miR-7 gene using PAMAM as the vector to determine the transfection efficiency and therapeutic effects invivo and invitro. We found that PAMAM exhibited higher transfection efficiency and longer duration of action compared with liposome delivery, and miR-7 efficiently silenced some genes involved in the epidermal growth factor receptor (EGFR) pathway and achieved favorable effects in treating glioma invivo and invitro. These investigations provide a basis for developing high-efficiency micromolecular drug delivery.

Current Status of Immunotherapy and Gene Therapy for High-Grade Gliomas

Despite improvements in surgical technique, radiation therapy delivery, and options for systemic cytotoxic therapy, the median survival for patients with newly diagnosed glioblastoma multiforme remains poor at 15 months with trimodality therapy. Multiple immunologic approaches are being tested to enhance the response of these tumors to existing therapy and/or to stimulate innate immune responses. We review the existing data that support the continued development of immunologic therapy in the treatment armamentarium against glioblastoma multiforme, with a focus on clinical data documenting outcomes. In phase I and phase II trials, antitumor vaccines (dendritic and formalin-fixed) have demonstrated clinical efficacy with mild toxicity, suggesting that innate immune responses can be amplified and directed against these tumors. Suicide gene therapy (gene-mediated cytotoxic therapy) using a number of viral vectors and molecular pathways has also shown efficacy in completed phase I and ongoing phase II trials. In addition, neural stem cells are being investigated as vectors in this approach. Although phase III data are needed before immunologic therapies can be widely implemented into clinical practice, the existing phase I and phase II data suggest that these therapies can produce meaningful and sometimes durable responses in patients with glioblastoma multiforme with mild toxicity compared with other existing therapies.

Whole-genome messenger RNA profiling reveals genes involved in malignant progression of glioma

To employ whole-genome messenger RNA profiling to identify the genes involved in malignant progression in glioma. The whole genome expressed genes were profiled in 220 glioma patients from the Chinese Glioma Genome Atlas (97 LGGs and 123 HGGs). The differential expressed genes between LGG and HGG were identified by SAM analysis. Microarray data were validated by immunohistochemistry. Among all the detected genes, the genes up-regulated mostly in high-grade glioma were IGFBP-2, CKLF, PTTG1, OSTCL and PTTG2 while those down-regulated mostly SEC31, RRP7B, HOOK3, SNRPN and CSMD3. Validation of IGFBP-2 with immunohistochemical staining showed a good correlation with the microarray data. A panel of potential genes of malignant transformation may serve as future targets of gene therapy for glioma.

Improved Adeno-associated Viral Gene Transfer to Murine Glioma

Glioblastoma (GBM) is a deadly primary brain tumor. Current treatment, consisting of surgical removal of the tumor mass followed by chemotherapy and/or radiotherapy, does not significantly prolong survival. Gene therapies for GBM are being developed in clinical trials, for example using adenoviral vectors. While adeno-associated virus (AAV) represents an alternative vector system, limited gene transfer to glioma cells has hampered its use. Here, we evaluated newly emerged variants of AAV capsid for gene delivery to murine glioma. We tested a mutant AAV2 capsid devoid of 3 surface-exposed tyrosine residues, AAV2 (Y444-500-730F), and a "shuffed" capsid (ShH19, containing sequences from several serotypes) that had previously been selected for enhanced glial gene delivery. AAV2 (Y-F) and ShH19 showed improved transduction of murine glioma GL261 cells in vitro by 2- to 6-fold, respectively, over AAV2. While AAV2 gene transfer to GL261 cells in established tumors in brains of syngeneic mice was undetectable, intratumoral injection of AAV2 (Y-F) or ShH19 resulted in local transduction of approximately 10% of tumor cells. In addition, gene transfer to neurons adjacent to the tumor was observed, while microglia were rarely transduced. Use of self-complementary vectors further increased transduction of glioma cells. Together, the data demonstrate the potential for improved AAV-based gene therapy for glioma using recently developed capsid variants.

Monitoring adenoviral based gene delivery in rat glioma by molecular imaging.

To determine whether endothelial progenitor cells (EPCs) can be used as delivery vehicle for adenoviral vectors and imaging probes for gene therapy in glioblastoma. To use cord blood derived EPCs as delivery vehicle for adenoviral vectors and imaging probes for glioma gene therapy, a rat model of human glioma was made by implanting U251 cells orthotopically. EPCs were transfected with an adenovirus (AD5/carrying hNIS gene) and labeled with iron oxide and inoculated them directly into the tumor 14 d following implantation of U251 cells. Magnetic resonance imaging (MRI) was used to in vivo track the migration of EPCs in the tumor. The expression of gene products was determined by in vivo Tc-99m single photon emission computed tomography (SPECT). The findings were validated with immunohistochemistry (IHC). EPCs were successfully transfected with the adenoviral vectors carrying hNIS which was proved by significantly (P < 0.05) higher uptake of Tc-99m in transfected cells. Viability of EPCs following transfection and iron labeling was not altered. In vivo imaging showed the presence of iron positive cells and the expression of transgene (hNIS) product on MRI and SPECT, respectively, all over the tumors following administration of transfected and iron labeled EPCs in the tumors. IHC confirmed the distribution of EPC around the tumor away from the injection site and also showed transgene expression in the tumor. The results indicated the EPCs' ability to deliver adenoviral vectors into the glioma upon intratumor injection. EPCs can be used as vehicle to deliver adenoviral vector to glioma and also act as imaging probe at the same time.

The glial fibrillary acidic protein promoter directs sodium/iodide symporter gene expression for radioiodine therapy of malignant glioma

Radioiodine is a routine therapy for differentiated thyroid cancers. Non-thyroid cancers may be treated with radio-iodine following transfection with the human sodium/iodide symporter (hNIS) gene. The glial fibrillary acidic protein (GFAP) promoter is an effective tumor-specific promoter for gene expression and thus may be useful in targeted gene therapy of malignant glioma. The present study used GFAP promoter-modulated expression of the hNIS gene in an experimental model of radioiodine-based treatment for malignant glioma. Cells were transfected using a recombination adeno-virus and evaluated in cells by studying the transfected transgene expression through western blot analysis, 125I uptake and efflux, clonogenicity following 131I treatment and radioiodine therapy using a U87 xenograft nude mouse model. Following transfection with the hNIS gene, the cells showed 95–70-fold higher 125I uptake compared with the control cells transfected with Ad-cytomegalovirus (CMV)-enhanced green fluorescent protein (EGFP). The western blotting revealed bands of ∼70, 49 and 43 kDa, consistent with the hNIS, GFAP and β-actin proteins. The clonogenic assay indicated that, following exposure to 500 μCi of 131I-iodide for 12 h, >90% of cells transfected with the hNIS gene were killed. Ad-GFAP-hNIS-transfected and 2 mCi 131I-injected U87 xenograft nude mice survived the longest of the three groups. The hNIS-expressing tumor tissue accumulated 99mTcO4 rapidly within 30 min of it being intraperitoneally injected. The experiments demonstrated that effective 131I therapy was achieved in the malignant glioma cell lines following the induction of tumor-specific iodide uptake activity by GFAP promoter-directed hNIS gene expression in vitro and in vivo. 131I therapy retarded Ad-GFAP-hNIS transfected-tumor growth following injection with 131I in U87 xenograft-bearing nude mice.

Valproic acid enhances anti-tumor effect of mesenchymal stem cell mediated HSV-TK gene therapy in intracranial glioma

Suicide gene therapy of glioma based on herpes simplex virus type I thymidine kinase (HSV-TK) and prodrug ganciclovir (GCV) suffers from the lack of efficacy in clinical trials, which is mostly due to low transduction efficacy and absence of bystander effect in tumor cells. Recently, stem cells as cellular delivery vehicles of prodrug converting gene has emerged as a new treatment strategy for malignant glioma. In this study, we evaluated the anti-glioma effect of suicide gene therapy using human bone marrow mesenchymal stem cells expressing HSV-TK (MSCs-TK) combined with valproic acid (VPA), which can upregulate the gap junction proteins and may enhance the bystander effect of suicide gene therapy. Expression of HSV-TK in MSCs was confirmed by RT-PCR analysis and the sensitivity of MSCs-TK to GCV was assessed. A bystander effect was observed in co-cultures of MSCs-TK and U87 glioma cells by GCV in a dose-dependent manner. VPA induced the expression of the gap junction proteins connexin (Cx) 43 and 26 in glioma cell and thereby enhanced the bystander effect in co-culture experiment. The enhanced bystander effect was inhibited by the gap junction inhibitor 18-β-glycyrrhetinic acid (18-GA). Moreover, the combined treatment with VPA and MSCs-TK synergistically enhanced apoptosis in glioma cells by caspase activation. In vivo efficacy experiments showed that combination treatment of MSCs-TK and VPA significantly inhibited tumor growth and prolonged the survival of glioma-bearing mice compared with single-treatment groups. In addition, TUNEL staining also demonstrated a significant increase in the number of apoptotic cells in the combination treated group compared with single-treatment groups. Taken together, these results provide the rational for designing novel experimental protocols to increase bystander killing effect against intracranial gliomas using MSCs-TK and VPA.

Rat Multipotent Mesenchymal Stromal Cells Lack Long-Distance Tropism to 3 Different Rat Glioma Models

Viral gene therapy of malignant brain tumors has been restricted by the limited vector distribution within the tumors. Multipotent mesenchymal stromal cells (MSCs) and other precursor cells have shown tropism for gliomas, and these cells are currently being explored as potential vehicles for gene delivery in glioma gene therapy. To investigate MSC migration in detail after intratumoral and extratumoral implantation through syngeneic and orthotopic glioma models. Adult rat bone marrow-derived MSCs were transduced to express enhanced green fluorescent protein and implanted either directly into or at a distance from rat gliomas. We found no evidence of long-distance MSC migration through the intact striatum toward syngeneic D74(RG2), N32, and N29 gliomas in the ipsilateral hemisphere or across the corpus callosum to gliomas located in the contralateral hemisphere. After intratumoral injection, MSCs migrated extensively, specifically within N32 gliomas. The MSCs did not proliferate within tumors, suggesting a low risk of malignant transformation of in vivo grafted cell vectors. Using a model for surgical glioma resection, we found that intratumorally grafted MSCs migrate efficiently within glioma remnants after partial surgical resection. The findings point to limitations for the use of MSCs as vectors in glioma gene therapy, although intratumoral MSC implantation provides a dense and tumor-specific vector distribution.

Over-expression of miR-145 enhances the effectiveness of HSVtk gene therapy for malignant glioma

This study attempts to combine two findings toward developing a rational strategy for improved therapy for glioma. One of the findings, made in this pre-clinical study, is that an hTERT-targeting ribozyme-controlled HSVtk gene (hTERT.Rz.HSVtk) exerts anti-tumor effects. The second observation is that the over-expression of a small noncoding RNA, miR-145, causes down-regulation of metastasis-related genes, such as PLAUR, SPOCK3, ADAM22, SLC7A5 and FASCN1. While blocking in vivo tumor growth only slightly, over-expression of miR-145 significantly inhibits both the migration and invasion of U87MG/U373MG glioma cells. We hypothesized that a simultaneous adenoviral-mediated over-expression of miR-145 might enhance the anti-tumor effects of hTERT.Rz.HSVtk and that a combination therapy with miR-145 and the HSVtk gene would be an effective approach for treating glioma. We tested this by developing adenoviral vectors that over-express miR-145 under the CMV promoter and employing them in combination with hTERT.Rz.HSVtk expression, both in vitro and in vivo in animal studies. We found that the adenovirus Ad5CMV.Rz.HSVtk.miR145 harboring an HSVtk expression cassette plus miR-145 produced prolonged survival benefits compared to administration of Ad5CMV.Rz.HSVtk or Ad5CMV.miR-145 alone. This study demonstrates that combination therapy using the hTERT.Rz.HSVtk gene together with miR-145 over-expression produces enhanced anti-tumor effects compared to that resulting from hTERT.Rz.HSVtk gene therapy alone.

Analysis of Wave Solutions of an Adhenovirus‐Tumor Cell System

We discuss biological background and mathematical analysis of glioma gene therapy for contributing to cancer treatment. By a reaction‐diffusion system, we model interactions between gliom cells and viruses. We establish some sufficient conditions on model parameters which guarantee the permanence of the system and the existence of periodic solutions. Our study has experimental and theoretical implication in the perspective management strategy of therapy.

The antitumor effect of mesenchymal stem cells transduced with a lentiviral vector expressing cytosine deaminase in a rat glioma model

Mesenchymal stem cells (MSCs) have been recognized as promising delivery vehicles for gene therapy of gliomas. The purpose of this study was to evaluate the antitumor effect of cytosine deaminase (CD)-expressing MSCs in a rat C6 glioma model. Lentiviral vectors expressing cytosine deaminase (CD) or enhanced green fluorescent protein (eGFP) were constructed and transduced into rat MSCs to generate MSC-CD/eGFP cells. By intracranially injecting C6 glioma cells (1 × 10(6)) alone or in combination with parental MSCs (1 × 10(6) or 2 × 10(6)) or MSC-CD/eGFP cells (1 × 10(6) or 2 × 10(6)) into rats, we examined the effect of engineered MSCs on tumor growth, tumor cell apoptosis, and rat survival in the presence of 5-fluorocytosine (5-FC). MSC-CD/eGFP cells were largely localized at the junction of the tumor with normal tissue. The mean survival time of rats co-injected with C6 glioma cells and MSC-CD/eGFP cells was significantly extended (C6 + MSC-CD/eGFP (1:1), 32.3 days; C6 + MSC-CD/eGFP (1:2), 45.9 days) when compared with rats injected with C6 glioma cells alone (15.3 days) or those co-injected with C6 glioma cells and parental cells (C6 + MSCs (1:1), 16.0 days; C6 + MSCs (1:2), 16.6 days). MSC-CD/eGFP-mediated gene therapy significantly reduced the tumor volume in C6 glioma-bearing rats. On day 14 after cell injection, the reduction in the mean tumor volume in rats co-injected with C6 + MSC-CD/eGFP cells (1:1 and 1:2) was 77.24 and 83.28%, respectively. In addition, MSC-CD/eGFP-mediated gene therapy promoted tumor cell apoptosis in rat C6 gliomas. Genetically engineered MSCs have good therapeutic efficacy against experimental gliomas in rats.

Complex based on Isthmin and RGD motif against glioma U251 cells

Objective To study the effect of complex based on Isthmin and RGD motif against glioma U251 cells.Methods The complex based on Isthmin and RGD motif was prepared,and the glioma U251 cell xenografts were established to observe the antitumor and antiangiogenesis effects of the complex in vivo and in vivo.Results The complex could induce U251 cell apoptosis and attack tumor endothelium cells,which inhibited the growth of glioma U251 cells and improved the lifespan of tumor bearing mice.Conclusion The complex based on Isthmin and RGD motif could dually target tumor and endothelium cells,which provided a promising strategy for glioma gene therapy. Key words: Isthmin; Glioma; Gene therapy

Clinical Development of Experimental Virus-Mediated Gene Therapy for Malignant Glioma

Advances in medical and surgical treatments in the last decades have resulted in quantum leaps in the overall survival of patients with many types of malignant disease, while survival of patients with malignant gliomas (WHO histological grades 3 and 4) has been only moderately improved. Maximum surgical resection, external fractionated radiotherapy, and oral chemotherapy during and after irradiation currently represent the pillars of malignant glioma therapy. Novel and experimental modalities aimed at a more selective and more effective treatment are however being increasingly developed and tested in clinical studies. Improved understanding of the fundamental mechanisms of glioma growth, resistance, and recurrence has resulted in the introduction of biologically and molecularly targeted therapies such as virus-mediated gene therapy, often in combination with spatially defined delivery methods specifically designed to be used in the local environment of the brain, such as convection-enhanced delivery. This review summarizes the key findings of the most important phase I and II clinical studies employing gene therapy with naturally occurring or genetically modified non-replicating or conditionally replicating (oncolytic) viruses, such as retrovirus, adenovirus, herpes-simplex-virus, Newcastle disease virus, or reovirus, in patients with primary or recurrent malignant gliomas. In addition, the two phase III gene therapy studies carried out to date in glioma patients and employing retrovirus or adenovirus vectors are presented in detail and critically discussed. Areas of necessary improvements and possible future developments of viruses and delivery methods are outlined.

Inhibition of C6 glioma in vivo by combination chemotherapy of implantation of polymer wafer and intracarotid perfusion of transferrin-decorated nanoparticles

The objective of this study was to develop a combination chemotherapy of implantation of a 3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-loaded wafer and intracarotid perfusion of BCNU-loaded nanoparticles for glioma treatment invivo. BCNU-loaded poly(D,L-lactic acid) (PLA) nanoparticles coated with transferrin (Tf) were prepared by a solvent evaporation/diffusion method using Tf as the emulsifier. X-ray photoelectron spectroscopy, Bratton-Marshall colorimetric assay and zeta-potential analysis confirmed the existence of Tf on the nanoparticles and their functional activities. BCNU-loaded PLA wafers were made of BCNU-loaded PLA microspheres. Invitro drug release behavior demonstrated that BCNU was released from the Tf-PLA nanoparticles and wafers in two distinct phases. The biodistribution of Tf-coated nanoparticles investigated by 99mTc-labeled single-photon emission computed tomography (SPECT) showed that the surface-containing Tf-PLA nanoparticles were concentrated in the brain. Inhibition of tumor growth in the C6 glioma-bearing animal model showed that combinational chemotherapy of BCNU-loaded wafer and BCNU-loaded PLA nanoparticles had a stronger inhibitory effect and prolonged the average survival time of rats (164%) compared with that of the control group. Furthermore, the tumors of this treatment group were not visible by examination at 4 weeks. The results of this study demonstrate for the first time that combination therapy of implantation of a BCNU-loaded wafer and intracarotid perfusion of BCNU-loaded nanoparticles may be a new strategy for glioma gene therapy.

Adenovirus-mediated delivery of bFGF small interfering RNA reduces STAT3 phosphorylation and induces the depolarization of mitochondria and apoptosis in glioma cells U251

Glioblastoma multiforme (GBM) carries a dismal prognosis primarily due to its aggressive proliferation in the brain regulated by complex molecular mechanisms. One promising molecular target in GBM is over-expressed basic fibroblast growth factor (bFGF), which has been correlated with growth, progression, and vascularity of human malignant gliomas. Previously, we reported significant antitumor effects of an adenovirus-vector carrying bFGF small interfering RNA (Ad-bFGF-siRNA) in glioma in vivo and in vitro. However, its mechanisms are unknown. Signal transducer and activator of transcription 3 (STAT3) is constitutively active in GBM and correlates positively with the glioma grades. In addition, as a specific transcription factor, STAT3 serves as the convergent point of various signaling pathways activated by multiple growth factors and/or cytokines. Therefore, we hypothesized that the proliferation inhibition and apoptosis induction by Ad-bFGF-siRNA may result from the interruption of STAT3 phosphorylation. In the current study, we found that in glioma cells U251, Ad-bFGF-siRNA impedes the activation of ERK1/2 and JAK2, but not Src, decreases IL-6 secretion, reduces STAT3 phosphorylation, decreases the levels of downstream molecules CyclinD1 and Bcl-xl, and ultimately results in the collapse of mitochondrial membrane potentials as well as the induction of mitochondrial-related apoptosis. Our results offer a potential mechanism for using Ad-bFGF-siRNA as a gene therapy for glioma. To our knowledge, it is the first time that the bFGF knockdown using adenovirus-mediated delivery of bFGF siRNA and its potential underlying mechanisms are reported. Therefore, this finding may open new avenues for developing novel treatments against GBM.

Vectors employed for malignant glioma gene therapy

The vectors play an important role in gene therapy by transporting the therapy genes into target tumor cells. At present there are mainly two kinds of gene vectors, including biological and non-biological vectors. Biological vectors, including virus, bacteria and stem cells, have good target activity and can efficiently delivery the genes into target tumor cells, but have potential genetic hazards. However, non-biological vectors, including liposomes and nanoparticles, can be simplely prepared with high genetic safety, but they can not transport the genes efficiently. Therefore, establishing safe and effective malignant glioma gene treatment system and finding gene vectors with good target activity become the research focus. Key words: Gliomas; Gene therapy; Genetic vectors

Telomerase reverse transcriptase promoter-driven expression of iodine pump genes for targeted radioiodine therapy of malignant glioma cells

Radioiodine is a routine therapy for differentiated thyroid cancers. Non-thyroid cancers can intake radioiodine after transfection of the human sodium iodide symporter (hNIS) gene. The human telomerase reverse transcriptase (hTERT) promoter, an excellent tumor-specific promoter, has potential value for targeted gene therapy of glioma. We used the hTERT promoter to drive the expression of the hNIS and human thyroid peroxidase (hTPO) gene as a primary step for testing the effects of radioiodine therapy on malignant glioma. The U87 and U251 cells were co-transfected with two adenoviral vectors, in which the hNIS gene had been coupled to the hTERT promoter and the hTPO gene had been coupled to the CMV promoter, respectively. Then, we performed Western blot, 125I intake and efflux assays, and clonogenic assay with cancer cells. We also did 99mTc tumor imaging of nude mice models. After co-transfection with Ad-hTERT-hNIS and Ad-CMV-hTPO, glioma cells showed the 125I intake almost 1.5 times higher than cells transfected with Ad-hTERT-hNIS alone. Western blots revealed bands of approximately 70 kDa and 110 kDa, consistent with the hNIS and hTPO proteins. In clonogenic assay, approximately 90% of cotransfected cells were killed, compared to 50% of control cells after incubated with 37 MBq of 131I. These results demonstrated that radioiodine therapy was effective in treating malignant glioma cell lines following induction of tumor-specific iodide intake by the hTERT promoter-directed hNIS expression in vitro. Cotransfected hNIS and hTPO genes can result in increased intake and longer retention of radioiodine. Nude mice harboring xenografts transfected with Ad-hTERT-NIS can take 99mTc scans.

Glioma Gene Therapy Using Induced Pluripotent Stem Cell Derived Neural Stem Cells

Using neural stem cells (NSCs) with tumor tropic migratory capacity to deliver therapeutic genes is an attractive strategy in eliminating metastatic or disseminated tumors. While different methods have been developed to isolate or generate NSCs, it has not been assessed whether induced pluripotent stem (iPS) cells, a type of pluripotent stem cells that hold great potential for regenerative medicine, can be used as a source for derivation of NSCs with tumor tropism. In this study, we used a conventional lentivirus transduction method to derive iPS cells from primary mouse embryonic fibroblasts and then generated NSCs from the iPS cells. To investigate whether the iPS cell derived NSCs can be used in the treatment of disseminated brain tumors, the cells were transduced with a baculoviral vector containing the herpes simplex virus thymidine kinase suicide gene and injected into the cerebral hemisphere contralateral to a tumor inoculation site in a mouse intracranial human glioma xenograft model. We observed that NSCs expressing the suicide gene were, in the presence of ganciclovir, effective in inhibiting the growth of the glioma xenografts and prolonging survival of tumor-bearing mice. Our findings provide evidence for the feasibility of using iPS cell derived NSCs as cellular vehicles for targeted anticancer gene therapy.

Inhibitory effect of knocking down miR-30a-5p on the growth of U251 glioma cells

Objective To investigate the effect of knock-down of miR-30a-5p on the biological characteristics of U251 glioblastoma cells. Methods miR-30a-5p inhibitor, mediated by Lipofectamine 2000, was transfected to U251 cells for knocking down miR-30a-5p. Real-time polymerase chain reaction (PCR) was conducted to detect the expression of miR-30a-5p in transfected cells. The cell proliferation rate was detected by methyl thiazol tetrazolium (MTT) assay, and cell cycle kinetics was examined by flow cytometry. The cell invasive ability was evaluated by Transwell assay and apoptosis detected by Annexin V assay. Results The expression of miR-30a-5p in the miR-30a-5p inhibitor group was significantly down-regulated. The cell proliferation activity and invasive ability were reduced. Cells were arrested in G0/G1 phase, and apoptosis was induced in cells transfected with miR-30a-5p inhibitor as compared to those of the cells transfected with scramble siRNA and control cells, so suppression of miR-30a-5p expression rendered the glioma cells harboring less aggressive phenotype. Conclusion miR-30a-5p is one of oncomiRs. It may be a candidate target miRNA for gene therapy of gliomas. Key words: Glioma; Proliferation; Invasion; Cell cycle