Differentiation and heterogeneity in T-antigen immortalized precursor cell lines from mouse cerebellum.
Recently, various techniques have been developed to transfer oncogenes into brain cells in order to generate immortalized neural cell lines. It is of interest to establish how well such cell lines reflect their cellular origin. Here we report the characterization of sixteen cell lines from mouse cerebellum and, as a control, six cell lines from skin. Lines were established by immortalizing postnatal primary cell cultures with a retrovirus carrying a modified temperature-sensitive variant of SV40 large T antigen. The cell lines reflect many properties of the cell type from which they were derived. All of the sixteen cerebellar lines expressed one or more markers of the neural precursor cells, namely, nestin and epitopes for NG2 and A2B5. In contrast, none of the six skin lines expressed neural precursor markers. Both types of cell lines expressed vimentin and fibronectin. Differentiation occurred in some of the cerebellar lines and was enhanced in defined medium. A small percentage of cerebellar cells, usually less than 5%, was positive for a marker of differentiation, e.g., glial fibrillary acidic protein (GFAP), galactocerebroside (GalC), or L1. Expression of GFAP colocalized with that of nestin at varying levels of intensity, indicating a gradual replacement of nestin by GFAP in the cytoskeleton. Both the cells positive for precursor markers and those positive for differentiation markers tended to be located in clusters, suggesting that stochastic processes or cell-cell interactions are important for the determination of the fate of cells within a clonal cell line in vitro. The degree of differentiation seemed to correlate with a shift from serum-containing to defined medium, but not with a shift from the permissive to the nonpermissive temperature for T antigen expression. The immortalization approach described here thus allows the establishment of cell lines which are "captured" in the precursor state of the developing mouse neuroepithelium.
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Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge.
Lendahl
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McKay
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D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000032:analysis / Q000235:genetics*; D002454:Cell Differentiation; D002455:Cell Division; D002461:Cell Line, Transformed; D002531:Cerebellum / Q000166:cytology*; D003470:Culture Media; D005814:Genes, Viral; D007158:Immunologic Techniques; D051379:Mice; D009857:Oncogenes; D013194:Staining and Labeling; D013234:Stem Cells / Q000166:cytology*; D013696:Temperature
D000952:Antigens, Polyomavirus Transforming; D003470:Culture Media