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Establishment of Immortal Neuronal Stem Cell Line from Embryonic Day 17 Rat Brain

(Journal of Korean Association of Cancer Prevention 2003; 8(4): 261-266)

Se-Ran Yang1, Sung-Dae Cho1, Nam-Shik Ahn1, Ji-Won Jung1, Joon-Suk Park1,Nguyen Ba Tiep1, Ki-Su Park1, In-Sun Hong1, Eun-Hye Jo1, Min-Su Seo1,Byong-Su Yoon2, Yong-Soon Lee1 and Kyung-Sun Kang1

1)Department of Veterinary Public Health, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea; 2)Department of Biology, College of Natural Science, Kyonggi University, Suwon, 442-760, Korea

We have recently characterized Neurosphere-derived cells from embryonic day (ED)17 rat fetus brain. Neural stem cells (NSCs) have been shown to reside in both fetal and adult brain leading to formation of cell clusters termed “neurospheres” and the neurosphere-derived cells can differentiate into neurons, oligodendrocytes and astrocytes.

In this study, we reported the establishment of neuronal stem cell line using SV40 large T antigen. The results show that the cells were acquired extended life span and constant increase at early (~30 cumulative population doubling level (cpdl)), middle (~60 cpdl), and late (~100 cpdl) passages. To characterize the established immortal cell, immunofluorescent staining was performed using GFAP antibody for astrocyte, NSE for neuron, and Nestin for neuronal stem cells. These results showed that the established immortal cell line expressed nestin. These results suggest that the established cell line might be a neuronal stem cells, and stem cells might be also major target cells for the carcinogenesis.

Key Words: Immortalization, Stem cells, SV40 Large T-antigen, Nestin, Neurosphere-derived cells

Differential Expressions of Gap Junction Proteins during Differentiation of Rat Neuronal Stem Cells

Journal of Korean Association of Cancer Prevention 2003

Se-Ran Yang1, Sung-Dae Cho1, Nam-Shik Ahn1, Ji-Won Jung1, Joon-Suk Park1, Nguyen Ba Tiep1, Ki-Su Park1, In-Sun Hong1, Eun-Hye Jo1, Min-Su Seo1, Byong-Su Yoon2, Yong-Soon Lee1 and Kyung-Sun Kang1

1Department of Veterinary Public Health College of Veterinary Medicine, Seoul National University, 151-742; 2Department of Biology, College of Natural Science, Kyonggi University, Suwon, 442-760, Korea Gap junctional intercellular communication (GJIC) plays a key role during development, process of tissue differentiation, and in maintenance of adult tissue homeostasis. Neural stem cells leading to formation of cell clusters termed "neurospheres", can differentiate into neurons, oligodendrocytes, and astrocytes. We investigated the expression levels and distribution of connexin43 (Cx43) and connexin32 (Cx32), abundant gap junctional protein in neural cells and in neurospheres isolated from rat fetus embryonic day (ED) 17. During differentiation of neurospheres, expression of Cx43 and 32 were increased time-dependently within 72 h, and then decreased at 7 day in western blot analysis. TPA-induced inhibition of GJIC was confirmed by decreased fluorescence by SL/DT assay, and induced hyperphosphorylation of Cx43 while no changes in Cx32 levels in western blot assay. Our results indicate that GJIC may be a crucial role in the differentiation of neuronal stem cell. And this GJIC can be inhibited by TPA through the hyperphosphorylation of Cx43.

Keywords: Neurosphere, Stem cells, Gap junction, Cx43, Cx32, Differentiation

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