Summary
We have established a multipotent clonal cell line, named MEB5, from embryonic mouse forebrains after the infection of a retrovirus carrying E7 oncogene of human papillomavirus type 16. MEB5 cells proliferated in serum-free, epidermal growth factor (EGF)-supplemented medium. They expressed markers for neural precursor cells (nestin, A2B5, and RC1) and did not express markers for neurons (class III β-tubulin), astrocytes (glial fibrillary acidic protein), and oligodendrocytes (galactocerebroside). MEB5 cells were stably maintained in an undifferentiated state with a diploid karyotype in the presence of EGF. When they were deprived of EGF, about 50% of the cells died due apoptosis within 24 h. The remaining cells differentiated into neurons, astrocytes, or oligodendrocytes within 2 wk. The newly developed cells with neuronal morphology were immunoreactive for γ-aminobutyric acid and exhibited neuronal electrophysiological properties. When MEB5 cells were treated with leukemia inhibitory for 7 d, they were induced to differentiate exclusively into astrocytes. These results inducate that MEB5 is a cell line with characteristics of EGF-dependent, multipotent neural precursor cells. This cell line should provide a good model system to study the mechanisms of survival, proliferation, and differentiation of the multipotent precursor cells in the central nervous system.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Ahmed, S.; Reynolds, B. A.; Weiss, S. BDNF enhances the differentiation but not the survival of CNS stem cell-derived neuronal precursors. J. Neurosci. 15:5765–5778; 1995.
Barres, B. A.; Hart, I. K.; Coles, H. S. R., et al. Cell death and control of cell survival in the oligodendrocyte lineage. Cell 70:31–46; 1992.
Bartlett, P. F.; Reid, H. H.; Bailey, K. A., et al. Immortalization of mouse neural precursor cells by the c-myc oncogene. Proc. Natl. Acad. Sci. USA 85:3255–3259; 1988.
Bernard, O.; Reid, H. H.; Bartlett, P. F. Role of the c-myc and the N-myc proto-oncogenes in the immortalization of neural precursors. J. Neurosci. Res. 24:9–20; 1989.
Cepko, C. Immortalization of neural cells via oncogene transduction. Trends Neurosci. 11:6–8; 1988.
Chalmers-Redman, R. M. E.; Priestley, T.; Kemp, J. A., et al. In vitro propagation and inducible differentiation of multipotential progenitor cells from human fetal brain. Neuroscience 76:1121–1128; 1997.
Davis, A. A.; Temple, S. A self-renewing multipotential stem cell in embryonic rat cerebral cortex. Nature 372:263–266; 1994.
Dyson, N.; Howley, P. M.; Munger, K., et al. The human papillomavirus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science 243:934–937; 1989.
Edwards, M. A.; Yamamoto, M.; Caviness, V. S., Jr. Organization of radial glia and related cells in the developing murine CNS: an analysis based upon a new monoclonal antibody marker. Neuroscience 36:121–144; 1990.
Frederiksen, K.; Jat, P. S.; Valtz, N., et al. Immortalization of precursor cells from the mammalian CNS. Neuron 1:439–448; 1988.
Gage, F. H.; Ray, J.; Fisher, L. J. Isolation, characterization, and use of stem cells from the CNS. Annu. Rev. Neurosci. 18:159–192; 1995.
Gritti, A.; Parati, E. A.; Cova, L., et al. Multipotential stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor. J. Neurosci. 16:1091–1100; 1996.
Inoue, H.; Kondoh, G.; Kamakura, C. R. et al. Progression of rat embryo fibroblast cells immortalized with transforming genes of human papillomavirus type 16. Virology 180:191–198; 1991.
Johe, K. K.; Hazel, T. G.; Muller, T., et al. Single factors direct the differentiation of stem cells from the fetal and adult central nervous system. Genes & Dev. 10:3129–3140; 1996.
Kilpatrick, T. J.; Richards, L. J.; Bartlett, P. F. The regulation of neural precursor cells within the mammalian brain. Mol. Cell. Neurosci. 6:2–15; 1995.
Lendahl, U.; McKay, D. G. The use of cell lines in neurobiology. Trends Neurosci. 13:132–137; 1990.
Lendahl, U.; Zimmerman, L. B.; McKay, R. D. G. CNS stem cells express a new class of intermediate filament protein. Cell 60:585–595; 1990.
Marone, M.; Quinones-Jenab, V.; Meiners, S., et al. An immortalized mouse neuroepithelial cell line with neuronal and glial phenotypes. Dev. Neurosci. 17:311–323; 1995.
Michler-Stuke, A.; Wolff, J. R.; Bottenstein, J. E. Factors influencing astrocyte growth and development in defined media. Int. J. Dev. Neurosci. 2:575–584; 1984.
Nakafuku, M.; Nakamura, S. Establishment and characterization of a multipotential neural cell line that can conditionally generate neurons, astrocytes, and oligodendrocytes in vitro. J. Neurosci. Res. 41:153–168; 1995.
Nakagaito, Y.; Yoshida, T.; Satoh, M., et al. Effects of leukemia inhibitory factor on the differentiation of astrocyte progenitor cells from embryonic mouse cerebral hemispheres. Dev. Brain Res. 87:220–223; 1995.
Raff, M. C.; Abney, E. R.; Cohen, J., et al. Two types of astrocytes in cultures of developing rat white matter: differences in morphology, surface gangliosides, and growth characteristics. J. Neurosci. 3:1289–1300; 1983.
Redies, C.; Lendahl, U.; McKay, R. D. G. Differentiation and heterogeneity in T-antigen immortalized precursor cell lines from mouse cerebellum. J. Neurosci. Res. 30:601–615; 1991.
Renfranz, P. J.; Cunningham, M. G.; McKay, R. D. G. Region-specific differentiation of the hippocampal stem cell line HiB5 upon implantation into the developing mammalian brain. Cell 66:713–729; 1991.
Reynolds, B. A.; Tetzlaff, W.; Weiss, S. A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. J. Neurosci. 12:4565–4574; 1992.
Reynolds, B. A.; Weiss, S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Nature 255:1707–1710; 1992.
Reynolds, B. A.; Weiss, S. Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev. Biol. 175:1–13; 1996.
Ryder, E. F.; Snyder, E. Y.; Cepko, C. L. Establishment and characterization of multipotent neural cell lines using retrovirus vector-mediated oncogene transfer. J. Neurobiol. 21:356–375; 1990.
Svendsen, C. N.; Fawcett, J. W.; Bentlage, C., et al. Increased survival of rat EGF-generated CNS precursor cells using B27 supplemented medium. Exp. Brain Res. 102:407–414; 1995.
Temple, S.; Davis, A. A. Isolated rat cortical progenitor cells are maintained in division in vitro by membrane-associated factors. Development 120:999–1008; 1994.
Visger, J. R. V.; Yeon, D. S.; Oh, T. H., et al. Differentiation and maturation of astrocytes derived from neuroepithelial progenitor cells in culture. Exp. Neurol. 128:34–40; 1994.
Ware, C. B.; Horowitz, M. C.; Renshaw, B. R., et al. Targeted disruption of the low-affinity leukemia inhibitory factor receptor gene causes placental, skeletal, neural and metabolic defects and results in perinatal death. Development 121:1283–1299; 1995.
Weiss, S.; Reynolds, B. A.; Vescovi, A. L., et al. Is there a neural stem cell in the mammalian forebrain? Trends Neurosci. 19:387–393; 1996.
Yoshida, T.; Satoh, M.; Nakagaito, Y., et al. Cytokines affecting survival and differentiation of an astrocyte progenitor cell line. Dev. Brain Res. 76:147–150; 1993.
Yoshida, T.; Takeuchi, M. Establishment of an astrocyte progenitor cell line: induction of glial fibrillary acidic protein and fibronectin by transforming growth factor-β1. J. Neurosci. Res. 35:129–137; 1993.
Yutsudo, M.; Okamoto, Y.; Hakura, A. Functional dissociation of transforming genes of human papillomavirus type 16. Virology 166:594–597; 1988.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Nakagaito, Y., Satoh, M., Kuno, H. et al. Establishment of an epidermal growth factor-dependent, multipotent neural precursor cell line. In Vitro Cell.Dev.Biol.-Animal 34, 585–592 (1998). https://doi.org/10.1007/s11626-998-0119-5
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/s11626-998-0119-5