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Characterization of astrocytes derived from human NTera‐2/D1 embryonal carcinoma cells
Author(s) -
Sandhu Jagdeep K.,
Sikorska Marianna,
Walker P. Roy
Publication year - 2002
Publication title -
journal of neuroscience research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.72
H-Index - 160
eISSN - 1097-4547
pISSN - 0360-4012
DOI - 10.1002/jnr.10236
Subject(s) - glutamate receptor , glutamate aspartate transporter , biology , astrocyte , glutamine synthetase , glial fibrillary acidic protein , glutamine , microbiology and biotechnology , glutamatergic , glutamic acid , biochemistry , metabotropic glutamate receptor , amino acid , endocrinology , immunohistochemistry , central nervous system , immunology , receptor
Astrocytes are the predominant cell type in the vicinity of glutamatergic synapses, where they monitor and maintain low levels of glutamate. Synaptic homeostasis of glutamate involves its removal from the synaptic cleft via high‐affinity glutamate transporters, glutamate transporter‐1 (GLT‐1)/excitatory amino acid transporters (EAAT)2 and glutamate and aspartate transporter (GLAST)/EAAT1, and glutamate‐catabolizing enzyme, glutamine synthase. Glutamate transporters have been mostly characterized in rodent astrocytes, due to the lack of a convenient human cell system. We report here that NTera‐2 (NT2/D1, a cell line derived from a human teratocarcinoma and known to differentiate into neurons) can also be differentiated by a 4‐week treatment with retinoic acid into functional astrocytes (NT2/A). Differentiation was accompanied by decreased cell proliferation and cell‐cycle arrest, as measured by flow cytometry, immunostaining for Ki67 and incorporation of 5‐bromo‐2′deoxyuridine (BrdU). Immunocytochemistry and Western blot analysis showed that NT2/A expressed glial fibrillary acidic protein, vimentin and S100β. Reverse transcription polymerase chain reaction (PCR) detected mRNA encoding glutamate transporters GLT‐1/EAAT2 and GLAST/EAAT1. The expression level of GLAST/EAAT1 was higher than that of GLT‐1/EAAT2, which is a typical expression pattern for primary astrocytes. Functionality of the transporters was demonstrated by the uptake of 3 H‐glutamate. NT2/A also expressed active glutamine synthase, and treatment with glutamate (up to 1 mM for 24 hr) was non‐toxic, suggesting that these cells were capable of converting it to non‐toxic metabolites. NT2/A and NT2‐derived neurons could be grown as mixed cultures and this may prove to be a useful experimental model to study molecular mechanisms underlying glutamate excitotoxicity. © 2002 Government of Canada. Exclusive worldwide publication rights in the article have been transferred to Wiley‐Liss, Inc.