• The Korean Journal of Physiology and Pharmacology
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• v.17 no.1
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• pp.23-30
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• 2013

Neural stem cells (NSCs) have the ability to proliferate and differentiate into various types of cells that compose the nervous system. To study functions of genes in stem cell biology, genes or siRNAs need to be transfected. However, it is difficult to transfect ectopic genes into NSCs. Thus to identify the suitable method to achieve high transfection efficiency, we compared lipid transfection, electroporation, nucleofection and retroviral transduction. Among the methods that we tested, we found that nucleofection and retroviral transduction showed significantly increased transfection efficiency. In addition, with retroviral transduction of Ngn2 that is known to induce neurogenesis in various types of cells, we observed facilitated final cell division in rat NSCs. These data suggest that nucleofection and retroviral transduction provide high efficiency of gene delivery system to study functions of genes in rat NSCs.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.19-19
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• 2002

This study was to examine whether the in vitro differentiated neural cells derived from human embryonic stem (hES, MB03) cells can be survived and expressed tyrosin hydroxylase(TH) in grafted normal or PD rat brain. To differentiate in vitro into neural cells, embryoid bodies (EB: for 5 days, without mitogen) were formed from hES cells, neural progenitor cells(neurosphere, for 7-10 days, 20 ng/㎖ of bFGF added N2 medium) were produced from EB, and then finally neurospheres were differentiated into mature neuron cells in N2 medium(without bFGF) for 2 weeks. In normal rat brain, neural progenitor cells or mature neuron cells (1×10/sup 7/ cells/㎖) were grafted to the striatum of normal rats. After 2 weeks, when the survival of grafted hES cells was examined by immunohistochemical analysis, the neural progenitor cell group indicated higher BrdU, NeuN＋, MAP2＋ and GFAP＋ than mature neuron cell group in grafted sites of normal rats. This result demonstrated that the in vivo differentiation of grafted hES cells be increased simultaneously in both of neuronal and glial cell type. Also, neural progenitor cell grafted normal rats expressed more TH pattern than mature neuron cells. Based on this data, as a preliminary test, when the neural progenitor cells were grafted into the striatum of 6-hydroxydopamine lesioned PD rats, we confirmed the cell survival (by double staining of Nissl and NeuN) and TH expression. This result suggested that in vitro differentiated neural progenitor cells derived from hES cells are more usable than mature neuron cells for the neural cell grafting in animal model and those grafted cells were survived and expressed TH in normal or PD rat brain.

• Biomedical Science Letters
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• v.13 no.4
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• pp.273-278
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• 2007

Stem cells have been the subject of increasing scientific interest because of their utility in numerous biomedical applications. Stem cells are capable of renewing themselves; that is, they can be continuously cultured in an undifferentiated state, giving rise to more specialized cells of the human body. Therefore, stem cells are an important new tools for developing unique, in vitro model systems to test drugs and chemicals and a potential to predict or anticipate toxicity in humans. In the present study, in vitro cultured F3 immortalized human neural stem cell line and in vivo adult Sprague Dawley rats was used to evaluate the cytotoxicity of anticancer drug paclitaxel. In vitro apoptotic activity of paclitaxel was evaluated in F3 cell line by a MTT assay and DAPI test. The cell death was induced with the treatment of 20 nM paclitaxel and chromatin degradation was detected by DAPI staining, which was analyzed by fluorescent microscope. In vivo studies, we also observed nestin immunoreactivity on subventricular zone, which is stem cell rich region in the adult brain of the SD rat. Immunofluorescent staining result shows that pixel intensities of nestin were decreased in a dose dependent manner. These results suggest that paclitaxel is able to induce cytotoxic activity both in F3 neural stem cell line and neural stem cell in SD rat brain.

• Biomolecules & Therapeutics
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• v.32 no.1
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• pp.65-76
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• 2024

Bortezomib (BTZ) is a proteasome inhibitor used to treat multiple myeloma (MM). However, the induction of peripheral neuropathy is one of the major concerns in using BTZ to treat MM. In the current study, we have explored the effects of BTZ (0.01-5 nM) on rat neural stem cells (NSCs). BTZ (5 nM) induced cell death; however, the percentage of neurons was increased in the presence of mitogens. BTZ reduced the B-cell lymphoma 2 (Bcl-2)/Bcl-2 associated X protein ratio in proliferating NSCs and differentiated cells. Inhibition of βIII-tubulin (TUBB3) degradation was observed, but not inhibition of glial fibrillary acidic protein degradation, and a potential PEST sequence was solely found in TUBB3. In the presence of growth factors, BTZ increased cAMP response element-binding protein (CREB) phosphorylation, brain-derived neurotrophic factor (Bdnf) transcription, BDNF expression, and Tubb3 transcription in NSCs. However, in the neuroblastoma cell line, SH-SY5Y, BTZ (1-20 nM) only increased cell death without increasing CREB phosphorylation, Bdnf transcription, or TUBB3 induction. These results suggest that although BTZ induces cell death, it activates neurogenic signals and induces neurogenesis in NSCs.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.4
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• pp.216-221
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• 2011

Objective: To differentiate the human embryonic stem cells (hESCs) into the retinal pigment epithelium (RPE) in the defined culture condition and determine its therapeutic potential for the treatment of retinal degenerative diseases. Methods: The embryoid bodies were formed from hESCs and attached on the matrigel coated culture dishes. The neural structures consisting neural precursors were selected and expanded to form rosette structures. The mechanically isolated neural rosettes were differentiated into pigmented cells in the media comprised of N2 and B27. Expression profiles of markers related to RPE development were analyzed by reverse transcription-polymerase chain reaction and immunostaining. Dissociated putative RPE cells ($10^5$ cells/5 ${\mu}L$) were transplanted into the subretinal space of rat retinal degeneration model induced by intravenous sodium iodate injection. Animals were sacrificed at 1, 2, and 4 weeks after transplantation, and immnohistochemistry study was performed to verify the survival of the transplanted cells. Results: The putative RPE cells derived from hESC showed characteristics of the human RPE cells morphologically and expressed molecular markers and associated with RPE fate. Grafted RPE cells were found to survive in the subretinal space up to 4 weeks after transplantation, and the expression of RPE markers was confirmed with immunohistochemistry. Conclusion: Transplanted RPE cells derived from hESC in the defined culture condition successfully survived and migrated within subretinal space of rat retinal degeneration model. These results support the feasibility of the hESC derived RPE cells for cell-based therapies for retinal degenerative disease.

• Journal of Life Science
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• v.12 no.3
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• pp.306-316
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• 2002

The ultrastructural change of sciatic nerve and immunohistochemical changes of NGF, PCNA were studied at the transplanted segment of intravascular cultured neural stem cell in the rat sciatic nerve by 5 months after the sciatic nerve transection. The transplanted intravascular neural stem cells were differentiated into Schwann reals at the 20th day and these cells began to regenerate by the proliferation and hypertrophy. There were many remyelinating Schwann cells in the transplanted nerve in term of stimulation. According to NGF finding, we suggest preexisting Schwann cells may induce the differentiation of neural stem cells into regenerating Schwann cells. Electron microscopic changes were the remyelinating appearance, the increase of intraaxonal microtubules and enlarged mitochondria and contacting tell processes each other.

• Journal of Oriental Neuropsychiatry
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• v.20 no.4
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• pp.63-77
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• 2009

Objectives : In this study, an essential oil fragrance from Danggwi was administrated into the neural stem cell and the effect of the essential oil on the differentiation and proliferation of the neural stem cells were observed. Methods : The establishment of the neural stem cell was identified via Nestin, DAPI dye. An essential oil fragrance from Danggwi was administrated with a proved optimum level for the survival of the cell through MTT assay. Also, according to the analysis of Western blot, the essential oil fragrance from Danggwi promotes the phosphorylating of Akt, Erk, ERM protein. Results : MTT assay showed increased in GFAP. The result indicates that the differentiation to astrocyte is promoted. The phosphorylation levels of ERM, Erk and Akt were increased at 60 min after addition of 5 ug/ml of essential oil fragrance from Danggwi and sustained to 48 hours. These imply that essential oil fragrance from Danggwi may induce the survival and the proliferation of the differentiated cells. Conclusions : These results suggest that the essential oil fragrance from Danggwi can be effective for the in vivo study of degenerative neuronal disease using neural stem cell.

• Biomolecules & Therapeutics
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• v.24 no.1
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• pp.99-107
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• 2016

Triclosan is an antimicrobial or sanitizing agent used in personal care and household products such as toothpaste, soaps, mouthwashes and kitchen utensils. There are increasing evidence of the potentially harmful effects of triclosan in many systemic and cellular processes of the body. In this study, we investigated the effects of triclosan in the survivability of cultured rat neural stem cells (NSCs). Cortical cells from embryonic day 14 rat embryos were isolated and cultured in vitro. After stabilizing the culture, triclosan was introduced to the cells with concentrations ranging from $1{\mu}M$ to $50{\mu}M$ and in varied time periods. Thereafter, cell viability parameters were measured using MTT assay and PI staining. TCS decreased the cell viability of treated NSC in a concentration-dependent manner along with increased expressions of apoptotic markers, cleaved caspase-3 and Bax, while reduced expression of Bcl2. To explore the mechanisms underlying the effects of TCS in NSC, we measured the activation of MAPKs and intracellular ROS. TCS at $50{\mu}M$ induced the activations of both p38 and JNK, which may adversely affect cell survival. In contrast, the activities of ERK, Akt and PI3K, which are positively correlated with cell survival, were inhibited. Moreover, TCS at this concentration augmented the ROS generation in treated NSC and depleted the glutathione activity. Taken together, these results suggest that TCS can induce neurodegenerative effects in developing rat brains through mechanisms involving ROS activation and apoptosis initiation.

• International Journal of Stem Cells
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• v.11 no.2
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• pp.177-186
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• 2018

Background and Objectives: Glial scarring and inflammation after spinal cord injury (SCI) interfere with neural regeneration and functional recovery due to the inhibitory microenvironment of the injured spinal cord. Stem cell transplantation can improve functional recovery in experimental models of SCI, but many obstacles to clinical application remain due to concerns regarding the effectiveness and safety of stem cell transplantation for SCI patients. In this study, we investigated the effects of transplantation of human mesenchymal stem cells (hMSCs) that were genetically modified to express Olig2 in a rat model of SCI. Methods: Bone marrow-derived hMSCs were genetically modified to express Olig2 and transplanted one week after the induction of contusive SCI in a rat model. Spinal cords were harvested 7 weeks after transplantation. Results: Transplantation of Olig2-expressing hMSCs significantly improved functional recovery in a rat model of contusive SCI model compared to the control hMSC-transplanted group. Transplantation of Olig2-expressing hMSCs also attenuated glial scar formation in spinal cord lesions. Immunohistochemical analysis showed that transplanted Olig2-expressing hMSCs were partially differentiated into Olig1-positive oligodendrocyte-like cells in spinal cords. Furthermore, NF-M-positive axons were more abundant in the Olig2-expressing hMSC-transplanted group than in the control hMSC-transplanted group. Conclusions: We suggest that Olig2-expressing hMSCs are a safe and optimal cell source for treating SCI.

• Reproductive and Developmental Biology
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• v.29 no.4
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• pp.229-234
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• 2005

Methods for activation of reconstructed oocytes were examined for the production of nuclear transfer (NT) rat embryos using fetal neural stem cells as donor. Neural stem cells were isolated from Day 14.5 rat fetuses, and the oocytes for recipient cytoplasm were recovered from 4-week old Sprague Dawley rats. After enucleation and nuclear injection, the reconstructed oocytes were immediately exposed to activation medium consisting of 10 mM $SrCl_2$ for 4 h (immediate activation after injection; IAI), or cultured in vitro for $2\~3$ h before activation treatment (injection before activation; IBA). Pre-activated oocytes were also used for NT to test reprogramming potential of artificially activated oocytes. The oocytes were grouped as IIA (immediate injection after activation) and ABI (activation $2\~3$ h before injection). Following NT, the oocytes were cultured in vitro. Development of the NT embryos was monitored at 44 and 119 h after activation. The embryos in groups IAI, mA, and IIA were cleaved to the 2-cell stage at the rates of $36.6\%\;(15/41),\;39.5\%\;(17/43)\;and\;46.3\%$ (25/54), respectively. However, in the ABI group, only one embryo ($1.8\%$, 1/55) was cleaved after activation. After in vitro culture, two NT embryos from IAI group had developed to the morula stage $(4.9\%\cdot2/41)$. However, no morula or blastocyst was obtained in the other groups. These results suggest that immediate activation after injection (IAI) method may be used for the production of rat somatic cell NT embryos.