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Darvishi M.,Tarbiat Modares University | Tiraihi T.,Tarbiat Modares University | Mesbah-Namin S.A.,Tarbiat Modares University | Delshad A.,Shahed University | Taheri T.,Shefa Neurosciences Research Center
Cellular and Molecular Neurobiology

Adipose-derived stem cells (ADSC) are adult stem cells which can be induced into motor neuron-like cells (MNLC) with a preinduction-induction protocol. The purpose of this study is to generate MNLC from neural stem cells (NSC) derived from ADSC. The latter were isolated from the perinephric regions of Sprague–Dawley rats, transdifferentiated into neurospheres (NS) using B27, EGF, and bFGF. After generating NSC from the NS, they induced into MNLC by treating them with Shh and RA, then with GDNF, CNTF, BDNF, and NT-3. The ADSC lineage was evaluated by its mesodermal differentiation and was characterized by immunostaining with CD90, CD105, CD49d, CD106, CD31, CD45, and stemness genes (Oct4, Nanog, and Sox2). The NS and the NSC were evaluated by immunostaining with nestin, NF68, and Neurod1, while the MNLC were evaluated by ISLET1, Olig2, and HB9 genes. The efficiency of MNLC generation was more than 95 ± 1.4 % (mean ± SEM). The in vitro generated myotubes were innervated by the MNLC. The induced ADSC adopted multipolar motor neuron morphology, and they expressed ISLET1, Olig2, and HB9. We conclude that ADSC can be induced into motor neuron phenotype with high efficiency, associated with differential expression of the motor neuron gene. The release of MNLC synaptic vesicles was demonstrated by FM1-43, and they were immunostained with synaptophysin. This activity was correlated with the intracellular calcium ion shift and membrane depolarization upon stimulation as was demonstrated by the calcium indicator and the voltage-sensitive dye, respectively. © 2016 Springer Science+Business Media New York Source

Oslau M.A.,Tarbiat Modares University | Tiraihi T.,Tarbiat Modares University | Hobbeneghi R.,Urmia University | Taheri T.,Shefa Neurosciences Research Center
Journal of Neurological Sciences

Selegiline has neuroprotective and antiapoptotic properties including anti-free radical and neurorophic factors induction. Hemisectioned spinal cord of rat was used to evaluate the effect of selegiline on the acute phase of spinal cord injury. Seven groups were used for studying the dose response, where 2.5 mg/kg (intraperitoneal) was selected as the best dose. Three groups were used for evaluating the neuroprotectivity, one group was sham-operated (SO) while the other two groups were subjected to spinal cord hemisection, which was done at T13 spinal segment level. One group (HT) was treated daily with 2.5 mg/kg selegiline for 21 days while the other was untreated group (HU). All groups were evaluated using Basso Beattie Bresnahan (BBB) behavioral test. Spinal tissues were processed, serially sectioned and stained with Cresyl violet. There were significant differences in the total motoneuron count (TMC) between HT and HU. Two indices were used to evaluate the improvement: the morphometric neuroprotection index and the recovery index (2.72. and 3.8, respectively). The recovery index was calculated using Gompertz model for the data of BBB scores in HT and HU, the maximum BBB scores were 13.75 and 9.33; the times of maximum response were 5.3 and 5.16; and the recovery rate coefficients were 20.6 and 5.32 for HT and HU, respectively. The conclusion is that selegiline is neuroprotective to the acute spinal cord injury. © 2014 Ege University Press. All rights reserved. Source

Albukhaty S.,Tarbiat Modares University | Naderi-Manesh H.,Tarbiat Modares University | Tiraihi T.,Shefa Neurosciences Research Center | Tiraihi T.,Tarbiat Modares University
Iranian Biomedical Journal

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 μg/ml SPION and in different concentrations of PLL (0.25, 0.50, 0.75, 1 and 2 μg/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 0.75μg/ml showed optimal results with 25 μg/ml SPION concentration compared with other PLL concentrations (0.25, 0.50, 1 and 2 μg/ml). The 18% efficiency with the transfected cells showed green fluorescence. Conclusion: Transfection with SPION is an efficient, non-viral gene transfere method. Source

Abdanipour A.,Tarbiat Modares University | Tiraihi T.,Tarbiat Modares University | Tiraihi T.,Shefa Neurosciences Research Center | Delshad A.,University of Tehran
Iranian Biomedical Journal

Background: Adult stem cells (ASC) are undifferentiated cells found throughout the body. These cells are promising tools for cell replacement therapy in neurodegenerative disease. Adipose tissue is the most abundant and accessible source of ASC. This study was conducted to evaluate effect of selegiline on differentiation of adiposederived stem cells (ADSC) into functional neuron-like cells (NLC), and also level of the neurotrophin expression in differentiated cells. Methods: ADSC were transdifferentiated into NLC using selegiline where CD90, CD49d, CD31, CD106 and CD45 were used as markers for ADSC identification. Lipogenic and osteogenic differentiation of ADSC were used to characterize the ADSC. ADSC were treated with selegiline at different concentrations (from 10-6 to 10-11 mM) and time points (3, 6, 12, 24 and 48 h). Percentage of viable cells, nestin and neurofilament 68 (NF-68) immunoreactive cells were used as markers for differentiation. The optimal dose for neurotrophin expressions in differentiating cells was evaluated using reverse transcriptase-PCR. NLC function was evaluated by loading and unloading with FM1-43 dye. Results: ADSC were immunoreactive to CD90 (95.67 ± 2.26), CD49d (71.52 ± 6.64) and CD31 (0.6 ± 0.86), but no immunoreactivity was detected for CD106 and CD45. The results of neural differentiation showed the highest percentage of nestin and NF-68 positive cells at 10-9 mM concentration of selegiline (exposed for 24 h). The differentiated cells expressed synapsin and neurotrophin genes except brainderived neurotrophic factor. Conclusion: ADSC can be an alternative source in cell-based therapy for neurodegenerative diseases using selegiline to induce ADSC differentiation to neuronal lineage. Source

Noori-Zadeh A.,Tarbiat Modares University | Mesbah-Namin S.A.,Tarbiat Modares University | Tiraihi T.,Tarbiat Modares University | Rajabibazl M.,Shahid Beheshti University of Medical Sciences | Taheri T.,Shefa Neurosciences Research Center
Journal of the Neurological Sciences

Glial cell line-derived neurotrophic factor (GDNF) is one of the most important proteins playing a pivotal role in growing and repairing of the nervous system. GDNF therapy is one of the suggested options in the treatment of neurodegenerative diseases. Limitations in the viral gene delivery and its side effects after therapy have encouraged us to use a non-viral method one for this purpose. We transfected rat bone marrow stromal cells (BMSCs) in ex vivo conditions using Lipofectamine 2000 reagent with pEGFP-C1 and a constructed vector carrying the human proGDNF (pSecTag2/HygroB-human proGDNF), transiently and stably, respectively. The rate of transient transfection of rat BMSCs was eight percent and transfected rat BMSCs with pSecTag2/HygroB-human proGDNF stabilized by adding Hygromycin B in cell culture medium at 200 μg/ml. Semi-quantitative data analysis from Western-blot technique showed that stable transfected cells secrete GDNF at higher level in comparison with control cells (6.530 fold in the supernatant). The present study supports the utility of liposome-mediated transfection for overexpressing human GDNF in rat BMSCs. For this purpose and in order to get more yield of human GDNF secretion from the stable transfected rat BMSCs, we used a vector containing another signal sequence instead of its own pre-segment of proGDNF protein. This is the first report in this regard and the data presented will be potentially useful for human gene transfer therapies in a variety of neurodegenerative diseases. © 2014 Elsevier B.V. Source

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