Background: The magnetic nanoparticle-based transfection method is a relatively new technique for delivery of functional genes to target tissues. We aimed to evaluate the transfection efficiency of rat neural stem cell (NSC) using poly-L-lysine hydrobromide (PLL) -coated super paramagnetic iron oxide nanoparticles (SPION).Methods: The SPION was prepared and coated with PLL as transfection agent and the transfection efficiency was evaluated in rat NSC using enhanced green fluorescent protein-N1 plasmid containing GFP as a reporter gene. NSC was incubated for 24 h in cell culture media containing 25 mg/ml SPION and in different concentrations of PLL (0.25, 0.50, 0.75, 1 and 2 mg/ml). Cell viability was determined before and after transfection for every concentration using Trypan blue assay. Characterization of prepared uncoated (SPION) and coated (SPION-PLL) complexes were evaluated by a transmission electron microscope and the zeta potential.Results: PLL at 0.75 mg/ml showed optimal results with 25 mg/ml SPION concentration compared with other PLL concentrations (0.25, 0.50, 1 and 2 mg/ml). The 18% efficiency of the transfected cells showed green fluorescence.Conclusion: Transfection with SPION is an efficient, non-viral gene transfere method.

Objective: The neuronal repressor REST (RE1-silencing traNSCription factor) is expressed at high levels in embryonic stem cells (ESC) with a critical role in self-renewal and pluripotency signaling network of these cells. REST is an essential element for brain development and also neuronal differentiation of ESC in vitro. Although REST is a well-known regulatory element in embryonic stem cells, but according to our knowledge, it has not been evaluated in induced pluripotent stem cell (iPSC) till now.Materials and Methods: The quantitative expression of REST in each step was evaluated by real-time PCR and the presence of REST was showed by immunofluorescent assay.Results: Q-PCR analysis showed that the expression level of REST decreased significantly during differentiation of hiPSCs to neural precursor cells, and remained stable until neuronal differentiation. immunofluorescent data revealed the both nuclear as cytoplasmic presence of REST in hiPSCs as well as NSCs, while in the maturated neurons it was only detectable in the cytoplasm.Conclusion: Our findings show a distinct expression pattern for REST during hESCs and hiPSCs neural differentiation. This study opens a new window for further experiments in this field to receive efficient differentiation of neuronal cells.

Objective: To evaluate the potential of Neural Stem Cell (NSC)-based approach to correct the metabolic defect and to ameliorate pathology in Twitcher (Twi) mice, a true model of Globoid Cell Leukodystrophy (GLD).Materials and Methods: Twi mice display severe demyelination and neurodegeneration, with a median survival of 46 days. We derived NSC lines from the subventricular zone of neonatal Twi mice and of wt littermates. Using bidirectional lentiviral vectors (bdLV) encoding for galactocerebrosidase (GALC) and GFP, we achieved high efficiency of transduction (>80%) and supraphysiological GALC levels (2-3 fold the wt levels) in mutant NSC, with no toxicity or functional impairment due to transgene over-expression. Similar supraphysiological GALC levels were obtained following bdLV. GALC transduction of wt NSC. Of importance, gene-corrected cells allowed more efficient metabolic cross-correction of GALC-deficient NSC then wt cells in vitro, due to more efficient enzyme secretion in the extracellular milieau. In order to obtain a stable source of GALC-secreting cells in the brain we transplanted wt or GALC over-expressing NSCs into the telencephalic lateral ventricles of neonatal (post-natal day 2) Twi mice (1x10^6 total cells, bilateral injection). Forty days after transplant we evaluated on NSC-treated mice and on untreated controls: i) NSC engraftment, distribution and fate (by immunocytochemistry); ii) the presence of a functional GALC protein in tissues and cerebrospinal fluid (by western blot and enzymatic assays); iii) intracellular storage (by lectin staining); iv) improvement of pathology (by immunocytochemistry and qPCR ). A group of treated mice was monitored for body weight and motor skills until a fixed human endpoint, in order to assess the impact of NSC therapy in delaying the onset of symptoms and prolonging lifespan.Results: Forty days after transplant we found NSC (identified by GFP expression) widely distributedinto the brain parenchyma, from the olfactory bulb to the hippocampus. Many cells were found lining the ventricles, along the corpus callosum and in the external capsula. Engrafted NSC (1-3% of the total injected cells) either expressed glial cell markers or retained antigenic features of immature neural cells and did not show proliferative activity. Of note, they robustly produced and secreted the GALC protein, as demonstrated by immunohistochemistry and by western blot using anti-GALC antibody. Most important, GALC activity was restored to 50% of wt levels in brain and spinal cord tissues of NSC-transplanted Twi mice, indicating widespread and efficient transport of the bioactive enzyme in CNS tissues (mainly through CSF flow) coupled to active cross-correction. The metabolic correction correlated with amelioration of pathology, clearance of tissue storage and partial rescue of the phenotype. We are currently evaluating the potential therapeutic advantage of GALC-overexpressing NSC as compared to the wt counterpart.Conclusion: These results, together with our preliminary data indicating the feasibility of safe GALC-overexpression in human fetal NSC (a therapeutically relevant cell type), warrant further consideration of NSC gene therapy for the treatment of GLD, likely in combination with other approaches (i.e. bone marrow transplant, currently under evaluation in our laboratory) ensuring enzymatic reconstitution in visceral organs and in the PNS.

Introduction: Ischemic stroke has high morbidity and mortality rates worldwide. Low oxygen (O2) levels detected in such conditions create a vulnerable environment for neural stem cells (NSC), altering neuronal function, and leading to neuronal injury or death. There are still no effective treatments for such consequences. This study investigates the molecular and functional effects of growth factors, namely, insulin-like growth factor 1 (IGF-I) and mechano growth factor (MGF), in NSC exposed to low O2 levels.  Methods: An in vitro ischemia model was created by rat hippocampal NSC grown in culture that was exposed to varying oxygen levels, including 0%, 3%, and 20 % for the representation of anoxic, hypoxic, and normoxic conditions, respectively, during 24 h. NSC has investigated IGF-I, MGF, and HIF1-Alpha (HIF-1α) gene expressions by real-time reverse traNSCription polymerase chain reaction. The effects of external administration of growth factors (IGF-I and MGF) on NSC proliferation in such conditions were explored.  Results: Increased IGF-I and MGF gene expressions were detected in the samples exposed to low O2. Anoxia was the highest stimulant for IGF-I and MGF gene expressions. Meanwhile, HIF1-α that encodes hypoxia-inducible factor-1α revealed downregulation in relative gene expression fold change with IGF-I application in all conditions, whereas MGF application upregulated its change in an anoxic environment. Furthermore, MGF-induced NSC had more proliferationmigration rate in all oxygen conditions. IGF-I induced significant NSC proliferation in 0% and 20% O2.  Conclusion: These findings suggest that IGF-I and MGF expressions were increased to reduce the damage in NSC exposed to low oxygen, and exogenous MGF and IGF-I application increased NSC proliferation at the time of injury. The results might imply the role of exogenous MGF and IGF-I in the treatment of ischemia for relieving the effect of neuronal damage due to their neuroprotective and proliferative effects.

Stroke-induced acute brain injury is a major cause of disability and mortality. Stem cell therapy has the ability to treat the neurological damage due to stroke worldwide. In vitro, Neural Stem Cell (NSC) stimulates axon growth and exhibit protective effects through the secretion of Nerve Growth Factor (NGF). The current study aims to determine the NGF levels in rats with hypoxic ischemia given neural stem cells. An animal experimental study was used. Sixteen healthy rats were divided into two groups, namely the hypoxia group and the hypoxia-treatment group. Both groups underwent the stroke model procedure. Hypoxia group was categorized as control group that received no intervention. The hypoxia-treatment group received Neural Stem Cell (NSC) intra-ventricularly. NSC was derived from Mesenchymal Stem Cell (MSC) adipose rat tissue. In the next 48 hours, the rats were then slowly sacrificed. The clinical proven stroke was assessed with neurological score. NGF levels were measured from blood serum and determined by ELISA examination. Positive expression of MSC was validated from CD 105, CD 90, and CD 29. Negative expressions of MSC were validated from CD 45. NSC was categorized by the Nestin expression. All rats in both groups suffered from hemiparesis with different levels based on neurology score evaluated on a six-point scale at 24 hours. There was statistically significant different NGF levels between groups after NSC procedure (p<0.05). The administered NSCs in stroke rat model produced higher levels of NGF.