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

Affiliations
  • 1Department of Pathology, Chonnam National University Medical School, Gwangju, Korea. mclee@jnu.ac.kr
  • 2Department of Forensic Medicine and Pathology, Chonnam National University Medical School, Gwangju, Korea.
  • 3Center for Biomedical Human Resources (BK 21), Chonnam National University Medical School, Gwangju, Korea.
  • 4Department of Pathology, Seonam University College of Medicine, Namwon, Korea.
  • 5Department of Ophthalmology, Chonnam National University Medical School, Gwangju, Korea.
  • 6Department of Physiology, Chonnam National University Medical School, Gwangju, Korea.
  • 7Department of Neurology, Chonnam National University Medical School, Gwangju, Korea.
  • 8Department of Pediatrics, Chonnam National University Medical School, Gwangju, Korea.
  • 9Department of Neurobiology, Chung-Ang University Medical Center, Seoul, Korea.
  • 10Department of Medicine, University of British Columbia, Vancouver, BC, Canada.

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

  • 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).

  • 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.

  • 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.

  • 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).

  • 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.


Reference

1. Metcalf D. Concise review: hematopoietic stem cells and tissue stem cells: current concepts and unanswered questions. Stem Cells. 2007. 25:2390–2395.
Article
2. Flax JD, Aurora S, Yang C, Simonin C, Wills AM, Billinghurst LL, Jendoubi M, Sidman RL, Wolfe JH, Kim SU, Snyder EY. Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes. Nat Biotechnol. 1998. 16:1033–1039.
3. Mimeault M, Batra SK. Recent progress on tissue-resident adult stem cell biology and their therapeutic implications. Stem Cell Rev. 2008. 4:27–49.
Article
4. Davis AA, Temple S. A self-renewing multipotential stem cell in embryonic rat cerebral cortex. Nature. 1994. 372:263–266.
Article
5. Kukekov VG, Laywell ED, Suslov O, Davies K, Scheffler B, Thomas LB, O'Brien TF, Kusakabe M, Steindler DA. Multipotent stem/progenitor cells with similar properties arise from two neurogenic regions of adult human brain. Exp Neurol. 1999. 156:333–344.
Article
6. Anderson DJ. Stem cells and pattern formation in the nervous system: the possible versus the actual. Neuron. 2001. 30:19–35.
7. Ourednik J, Ourednik V, Lynch WP, Schachner M, Snyder EY. Neural stem cells display an inherent mechanism for rescuing dysfunctional neurons. Nat Biotechnol. 2002. 20:1103–1110.
Article
8. Ryu JK, Kim J, Cho SJ, Hatori K, Nagai A, Choi HB, Lee MC, Mc-Larnon JG, Kim SU. Proactive transplantation of human neural stem cells prevents degeneration of striatal neurons in a rat model of Huntington disease. Neurobiol Dis. 2004. 16:68–77.
Article
9. Davidovics Z, DiCicco-Bloom E. Moderate lead exposure elicits neurotrophic effects in cerebral cortical precursor cells in culture. J Neurosci Res. 2005. 80:817–825.
Article
10. Breier JM, Radio NM, Mundy WR, Shafer TJ. Development of a high-throughput screening assay for chemical effects on proliferation and viability of immortalized human neural progenitor cells. Toxicol Sci. 2008. 105:119–133.
Article
11. Satoh J, Tabira T, Kim SU. Rapidly proliferating glial cells isolated from adult mouse brain have a differentiative capacity in response to cyclic AMP. Neurosci Res. 1994. 20:175–184.
Article
12. Rey JA, Bello MJ, De Campos JM, Kusak ME, Moreno S. Cytogenetic analysis in human neurinomas. Cancer Genet Cytogenet. 1987. 28:187–188.
Article
13. Kim SU, Stern J, Kim MW, Pleasure DE. Culture of purified rat astrocytes in serum-free medium supplemented with mitogen. Brain Res. 1983. 274:79–86.
Article
14. Cai J, Wu Y, Mirua T, Pierce JL, Lucero MT, Albertine KH, Spangrude GJ, Rao MS. Properties of a fetal multipotent neural stem cell (NEP cell). Dev Biol. 2002. 251:221–240.
Article
15. Nagai A, Suzuki Y, Baek SY, Lee KS, Lee MC, McLarnon JG, Kim SU. Generation and characterization of human hybrid neurons produced between embryonic CNS neurons and neuroblastoma cells. Neurobiol Dis. 2002. 11:184–198.
Article
16. Lee MC, Chung YT, Lee JH, Jung JJ, Kim HS, Kim SU. Antioxidant effect of melatonin in human retinal neuron cultures. Exp Neurol. 2001. 172:407–415.
Article
17. Bignami A, Raju T, Dahl D. Localization of vimentin, the nonspecific intermediate filament protein, in embryonal glia and in early differentiating neurons. In vivo and in vitro immunofluorescence study of the rat embryo with vimentin and neurofilament antisera. Dev Biol. 1982. 91:286–295.
18. Lendahl U, Zimmerman LB, McKay RD. CNS stem cells express a new class of intermediate filament protein. Cell. 1990. 60:585–595.
Article
19. Zhong W, Jiang MM, Weinmaster G, Jan LY, Jan YN. Differential expression of mammalian Numb, Numblike and Notch1 suggests distinct roles during mouse cortical neurogenesis. Development. 1997. 124:1887–1897.
Article
20. Andrae J, Hansson I, Afink GB, Nister M. Platelet-derived growth factor receptor-alpha in ventricular zone cells and in developing neurons. Mol Cell Neurosci. 2001. 17:1001–1013.
21. Kageyama R, Ohtsuka T. The Notch-Hes pathway in mammalian neural development. Cell Res. 1999. 9:179–188.
Article
22. Yang X, Handler M, Shen J. Role of presenilin-1 in murine neural development. Ann N Y Acad Sci. 2000. 920:165–170.
Article
23. Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med. 1996. 183:1797–1806.
Article
24. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science. 1999. 284:143–147.
Article
25. Martinez-Serrano A, Lundberg C, Horellou P, Fischer W, Bentlage C, Campbell K, McKay RD, Mallet J, Bjorklund A. CNS-derived neural progenitor cells for gene transfer of nerve growth factor to the adult rat brain: complete rescue of axotomized cholinergic neurons after transplantation into the septum. J Neurosci. 1995. 15:5668–5680.
Article
26. Rietze RL, Valcanis H, Brooker GF, Thomas T, Voss AK, Bartlett PF. Purification of a pluripotent neural stem cell from the adult mouse brain. Nature. 2001. 412:736–739.
Article
27. Ohkawara B, Okuno M, Ishii T, Horiuchi M, Tomooka Y. Characterization of a multipotent neural progenitor cell line cloned from an adult p53-/- mouse cerebellum. Brain Res. 2003. 959:11–19.
Article
28. Coyle JT, Puttfarcken P. Oxidative stress, glutamate, and neurodegenerative disorders. Science. 1993. 262:689–695.
Article
29. Shivakumar BR, Kolluri SV, Ravindranath V. Glutathione and protein thiol homeostasis in brain during reperfusion after cerebral ischemia. J Pharmacol Exp Ther. 1995. 274:1167–1173.
Full Text Links
  • JKMS
Actions
Cited
CITED
export Copy
Close
Share
  • Twitter
  • Facebook
Similar articles
Copyright © 2024 by Korean Association of Medical Journal Editors. All rights reserved.     E-mail: koreamed@kamje.or.kr