J Korean Med Sci.  2010 Mar;25(3):440-448. 10.3346/jkms.2010.25.3.440.

Neurotoxicity Screening in a Multipotent Neural Stem Cell Line Established from the Mouse Brain

• Choi YS
• Lee MC
• Kim HS
• Lee KH
• Park YG
• Kim HK
• Jeong HS
• Kim MK
• Woo YJ
• Kim SU
• Ryu JK
• Choi HB

• KMID: 1778041
• DOI: http://doi.org/10.3346/jkms.2010.25.3.440

Abstract

Neural stem cells (NSCs) have mainly been applied to neurodegeneration in some medically intractable neurologic diseases. In this study, we established a novel NSC line and investigated the cytotoxic responses of NSCs to exogenous neurotoxicants, glutamates and reactive oxygen species (ROS). A multipotent NSC line, B2A1 cells, was established from long-term primary cultures of oligodendrocyte-enriched cells from an adult BALB/c mouse brain. B2A1 cells could be differentiated into neuronal, astrocytic and oligodendroglial lineages. The cells also expressed genotypic mRNA messages for both neural progenitor cells and differentiated neuronoglial cells. B2A1 cells treated with hydrogen peroxide and L-buthionine-(S,R)-sulfoximine underwent 30-40% cell death, while B2A1 cells treated with glutamate and kainate showed 25-35% cell death. Cytopathologic changes consisting of swollen cell bodies, loss of cytoplasmic processes, and nuclear chromatin disintegration, developed after exposure to both ROS and excitotoxic chemicals. These results suggest that B2A1 cells may be useful in the study of NSC biology and may constitute an effective neurotoxicity screening system for ROS and excitotoxic chemicals.

Keyword

Growth Factors; Brain; Nestin; Stem Cells; Culture

MeSH Terms

Animals
Brain/*cytology
Buthionine Sulfoximine/pharmacology
Cell Differentiation
Cell Line
Cell Lineage
Cytokines/pharmacology
Enzyme Inhibitors/pharmacology
Excitatory Amino Acid Agonists/pharmacology
Glutamic Acid/pharmacology
Humans
Hydrogen Peroxide/pharmacology
Intercellular Signaling Peptides and Proteins/pharmacology
Kainic Acid/pharmacology
Mice
Mice, Inbred BALB C
Multipotent Stem Cells/cytology/*drug effects/physiology
Neuroglia/cytology/drug effects/physiology
Neurons/cytology/*drug effects/physiology
Neurotoxins/*pharmacology
Oxidants/pharmacology
Phenotype
Reactive Oxygen Species/metabolism
Cytokines
Enzyme Inhibitors
Excitatory Amino Acid Agonists
Intercellular Signaling Peptides and Proteins
Neurotoxins
Oxidants
Reactive Oxygen Species
Kainic Acid
Buthionine Sulfoximine
Glutamic Acid
Hydrogen Peroxide

Figure

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  Fig. 1 Cytologic characteristics of B2A1 cells include bi- or tripolar cells (A, phase contrast microscopy), which are immunopositive for nestin (B), β tubulin III (C), and GFAP (D) (×200).

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  Fig. 2 Multipotential differentiation of B2A1 cells by treatment with growth factors. Most cells were immunopositive for nestin in any culture condition (A). Cells immunopositive for β tubulin III significantly increased in RA-containing media (B), while GFAP (C) and GalC (D) positive cells increased markedly in c-AMP or PDGF-containing media.

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  Fig. 3 Gene expression of cell type specific markers studied by RT-PCR in B2A1 cells. The cells expressed both neural progenitor cell markers (nestin, Notch1, PDGFR-α) and differentiated cell markers for neurons (NF-L), astrocytes (GFAP), and oligodendrocytes (MBP). The expression levels vary depending upon the level of growth factors added.

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  Fig. 4 Cytopathologic features of B2A1 cells. Compared with the control cells (A), cells treated with BSO (10 µM) show swelling of cell bodies with axon hillocks, vacuolar changes with increased granularity of the cytoplasm at 4 hr (B), loss of neurites, progressive disintegration of nuclear chromatin with homogenization, and loss of cell to cell contact at 24 hr (C). Glutamate or kainate-treated cells (20 µM) also show swelling of cell bodies and loss of neurites at 12 hr (D) (×200).

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  Fig. 5 Effect of H2O2 (A), BSO (B), glutamate (C) and kainate (D) treatment for 24 hr, measured by the MTT test. Data are shown as mean±standard deviation. The number of viable cells are significantly decreased after the addition of 500 µM H2O2 along the stepwise increase of doses. However, a dose-response pattern is not observed in the BSO, glutamate, or kainite treated groups.

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