Combination Therapy by Tissue-Specific Suicide Gene and Bevacizumab in Intramedullary Spinal Cord Tumor

Purpose: Malignant gliomas are aggressive spinal cord tumors. In this study, we hypothesized that combination therapy using an anti-angiogenic agent, bevacizumab, and hypoxia-inducible glioblastoma-specific suicide gene could reduce tumor growth. Materials and methods: In the present study, we evaluated the effect of combination therapy using bevacizumab and pEpo-NI2-SV-TK in reducing the proliferation of C6 cells and tumor growth in the spinal cord. Spinal cord tumor was generated by the injection of C6 cells into the T5 level of the spinal cord. Complexes of branched polyethylenimine (bPEI)/pEpo-NI2-SV-TK were injected into the spinal cord tumor. Bevacizumab was then administered by an intraperitoneal injection at a dose of 7 mg/kg. The anti-cancer effects of combination therapy were analyzed by histological analyses and magnetic resonance imaging (MRI). The Basso, Beattie and Bresnahan scale scores for all of the treatment groups were recorded every other day for 15 days to assess the rat hind-limb strength. Results: The complexes of bPEI/pEpo-NI2-SV-TK inhibited the viability of C6 cells in the hypoxia condition at 5 days after treatment with ganciclovir. Bevacizumab was decreased in the cell viability of human umbilical vein endothelial cells. Combination therapy reduced the tumor size by histological analyses and MRI. The combination therapy group showed improved hind-limb function compared to the other groups that were administered pEpo-NI2-SV-TK alone or bevacizumab alone. Conclusion: This study suggests that combination therapy using bevacizumab with the pEpo-NI2-SV-TK therapeutic gene could be useful for increasing its therapeutic benefits for intramedullary spinal cord tumors.ope

Therapeutic Use of 3β-[N-(N',N'-Dimethylaminoethane) Carbamoyl] Cholesterol-Modified PLGA Nanospheres as Gene Delivery Vehicles for Spinal Cord Injury

Gene delivery holds therapeutic promise for the treatment of neurological diseases and spinal cord injury. Although several studies have investigated the use of non-viral vectors, such as polyethylenimine (PEI), their clinical value is limited by their cytotoxicity. Recently, biodegradable poly (lactide-co-glycolide) (PLGA) nanospheres have been explored as non-viral vectors. Here, we show that modification of PLGA nanospheres with 3β-[N-(N',N'-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) enhances gene transfection efficiency. PLGA/DC-Chol nanospheres encapsulating DNA were prepared using a double emulsion-solvent evaporation method. PLGA/DC-Chol nanospheres were less cytotoxic than PEI both in vitro and in vivo. DC-Chol modification improved the uptake of nanospheres, thereby increasing their transfection efficiency in mouse neural stem cells in vitro and rat spinal cord in vivo. Also, transgene expression induced by PLGA nanospheres was higher and longer-lasting than that induced by PEI. In a rat model of spinal cord injury, PLGA/DC-Chol nanospheres loaded with vascular endothelial growth factor gene increased angiogenesis at the injury site, improved tissue regeneration, and resulted in better recovery of locomotor function. These results suggest that DC-Chol-modified PLGA nanospheres could serve as therapeutic gene delivery vehicles for spinal cord injury.ope

Ctbp2와 결합하는 zinc finger protein, Zfp217과 Zfp516에 의한 배아줄기세포 분화 조절

학위논문 (박사)-- 서울대학교 대학원 : 의과대학 의과학과, 2018. 2. 윤홍덕.Transcription factors and chromatin remodeling proteins control the transcriptional variability for embryonic stem cell (ESC) lineage commitment. During ESC differentiation, chromatin modifiers are recruited to the regulatory regions by transcription factors, thereby activating the lineage-specific genes or silencing active ESC genes. On differentiation cue, active ESC genes are silenced by C-terminal binding protein 2 (Ctbp2) and its binding complexes, the nucleosome remodeling and deacetylation (NuRD) complex and the polycomb repressive complex 2 (PRC2), which each modify H3K27 deacetylation and tri-methylation mediated repression. However, Ctbp2 itself is unable to bind to the DNA, which leaves a missing link in Ctbp2-mediated epigenetic regulation during stem cell differentiation. Also, the underlying mechanisms that link transcription factors to the exit from pluripotency are not yet identified. Thus, it is necessary to search for transcription factors that fine-tune Ctbp2 and these epigenetic regulators on active ESC genes. In this study, we analyzed the role of the Ctbp2-interacting zinc finger proteins, Zfp217 and Zfp516, as linkers for the chromatin regulators during ESC differentiation. CRISPR-Cas9-mediated double knock-outs of both Zfp217 and Zfp516 in ESCs prevented the exit from pluripotency. Both zinc finger proteins cooperatively regulated the Ctbp2-mediated NuRD complex and PRC2 complex recruitment to active ESC genes, subsequently switching the H3K27ac to H3K27me3 during ESC differentiation. Therefore, we suggest that zinc finger proteins, Zfp217 and Zfp516, orchestrate with Ctbp2 to control the concise epigenetic states on active ESC genes during differentiation, resulting in natural lineage commitment.I. INTRODUCTION 1 1-1. Embryonic stem cell maintenance and development 2 1-2. Transcription factors function in ESC differentiation 3 1-3. C-terminal binding protein 2-mediated repression 4 1-4. Ctbp2-associated zinc finger proteins, Zfp217 and Zfp516 5 1-5. Purpose 7 II. MATERIALS AND METHODS 9 2-1. Cell culture 10 2-2. In vitro differentiation of ESCs 10 2-3. Self-renewal assay 10 2-4. Generation of zinc finger protein antibodies 11 2-5. Lentiviral shRNA-mediated knockdown 11 2-6. Genome editing with CRISPR/Cas9 12 2-7. Plasmids constructs 13 2-8. Generation of stable ESC lines 13 2-9. Immunoprecipitation and Western blot 14 2-10. Chromatin Immunoprecipitation (ChIP) assay 15 2-11. Real-time qPCR 17 2-12. Statistics 17 2-13. ChIP-seq data analysis 17 2-14. RNA-seq data analysis 18 III. Results 23 3-1. Ctbp2 interacts with zinc finger proteins in mouse ESCs 24 3-2. Zfp217 depletion retards ESC differentiation 35 3-3. Zfp217 and Ctbp2 are co-localized at actively transcribed regions in ESCs 48 3-4. Zfp217 associates with repressive chromatin modifiers on active ESC genes 55 3-5. Synergistic effect of Ctbp2-associated zinc finger proteins on ESC differentiation 67 3-6. Zfp217 and Zfp516 facilitate Ctbp2-mediated repression on active ESC genes during differentiation 76 IV. DISCUSSION 85 V. CONCLUSION 90Docto

Multifunctional nanoparticles for gene delivery and spinal cord injury

Methylprednisolone (MP) is a glucocorticoid that is used as an anti-inflammatory agent to the treat spinal cord injury (SCI). A low molecular weight chitosan was used to synthesize chitosan-MP conjugate, which was used to evaluate the gene therapy, anti-inflammatory and anti-apoptotic effects of MP. The cytotoxicity of chitosan-MP nanoparticles and the transfection efficiency of plasmid DNA were evaluated by MTT and luciferase assays. A chitosan-MP/pDNA complexes was injected into injured spinal cord to evaluate the anti-inflammatory and anti-apoptotic effects of these complexes using terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) and ED1 staining, respectively. In addition, to evaluate the distribution of chitosan-MP/pDNA complexes, pβ-gal encapsulated chitosan-MP was injected into the injected site. Cell survival was similar in cells treated with chitosan-MP conjugate and untreated cells. Luciferase expression was higher in cells treated with the chitosan-MP/pDNA than cells treated with the chitosan/pDNA. The chitosan-MP/pDNA complexes also reduced apoptosis and inflammation at the injury site. These results suggest that chitosan-MP conjugation is an effective gene delivery system to treat SCI. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 3474-3482, 2015.ope

Combined therapy using embryonic stem cell transplantation and cytokine administration for treatment of myocardial infarction

In the present study, we tested the hypothesis that granulocyte colony-stimulating factor (G-CSF) administration would enhance the efficacy of cellular cardiomyoplasty with embryonic stem (ES) cells in infarcted myocardium. Three weeks after myocardial infarction by cryoinjury, Sprague-Dawley rats were randomized to receive either an injection of medium, ES cell transplantation, G-CSF administration, or a combination of G-CSF administration and ES cell transplantation. Prior to transplantation, ES cells were induced to differentiate into cardiomyocytes. Eight weeks after treatment, the cardiac tissue formation, neovascularization, and apoptotic activity in the infarct regions were evaluated by histology and immunohistochemistry. The left ventricular (LV) dimensions and function of the treated heart were evaluated by echocardiography. Transplanted ES cells survived and participated in the myocardial regeneration in the infarcted heart. A combination of G-CSF treatment and ES cell transplantation significantly promoted angiogenesis and reduced the infarct area and cell apoptosis in the infarcted myocardium compared with ES cell transplantation alone. The combination therapy also attenuated LV dilation, ascompared with ES cell transplantation alone. The present study demonstrates that G-CSF treatment can enhance the efficacy of cellular cardiomyoplasty by ES cell transplantation to treat myocardial infarction.N

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.ope

Effect of Combined Bevacizumab and Temozolomide Treatment on Intramedullary Spinal Cord Tumor

STUDY DESIGN: C6 glioma cells and an intramedullary spinal cord tumor model were used to evaluate the effect of bevacizumab (Avastin) or temozolomide (TMZ). OBJECTIVE: In this study, we hypothesized that treatment with bevacizumab accelerates the therapeutic effect of TMZ on intramedullary gliomas in an animal model. SUMMARY OF BACKGROUND DATA: Recently therapies for the management of intramedullary malignant gliomas include surgery, chemotherapy, and radiotherapy. Concurrent or adjuvant TMZ has been considered an emerging new treatment for intramedullary malignant gliomas; however, high-dose application of TMZ has limitation of side effect. METHODS: C6 glioma cells were injected into the T5 level of the spinal cord, and TMZ and bevacizumab were administered 5 days after C6 inoculation (n = 7 for each group). Tumor size was analyzed using histology and magnetic resonance imaging at 13 days after tumor inoculation. RESULTS: Histological analyses and magnetic resonance imaging findings showed that combined treatment with TMZ and bevacizumab reduced tumor mass. The tumor volume of control group was 2.8-fold higher than combined therapy (P < 0.05). Neurological outcomes demonstrated that combined therapy improved hind limb function more than TMZ-alone group or control group (P < 0.05). CONCLUSION: This study shows that bevacizumab could be useful in combination with TMZ to increase the therapeutic benefits of TMZ for intramedullary spinal cord tumors. LEVEL OF EVIDENCE: N/A.ope

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 (H(2)O(2)), and combined (SD + H(2)O(2)) injury mimicking the ischemic environment of the injured spinal cord. We demonstrated that AT-MSCs inhibited the apoptosis of mNSCs in SD, H(2)O(2), 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.ope