A novel human astrocyte cell line (A735) with astrocyte-specific neurotransmitter function.
Studies of brain cell function and physiology are hampered by the limited availability of immortal human brain-derived cell lines, as a result of the technical difficulties encountered in establishing immortal human cells in culture. In this study, we demonstrate the application of recombinant DNA vectors expressing SV40 T antigen for the development of immortal human cell cultures, with morphological, growth, and functional properties of astrocytes. Primary human astrocytes were transfected with the SV40 T antigen expression vectors, pSV3neo or p735.6, and cultures were established with an extended lifespan. One of these cultures gave rise to an immortal cell line, designated A735. All the human SV40-derived lines retained morphological features and growth properties of type 1 astrocytes. Immunohistochemical studies and Western blot analysis of the intermediate filament proteins and glutamine synthetase demonstrated a differentiated but immature astrocyte phenotype. Transport of gamma-amino butyric acid and glutamate were examined and found to be by a glial-specific mechanism, consistent with the cell lines' retaining aspects of normal glial function. We conclude that methods based on the use of SV40 T antigen can successfully immortalize human astrocytes, retaining key astrocyte functions, but T antigen-induced proliferation appeared to interfere with expression of glial fibrillary acidic protein. We believe A735 is the first documented nontumor-derived human glial cell line which is immortal.
Price
T N
TN
Trafford Centre for Graduate Medical Education and Research, Division of Molecular Medicine, University of Sussex, Falmer Brighton, United Kingdom.
Burke
J F
JF
Mayne
L V
LV
D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D001253:Astrocytes / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D015153:Blotting, Western; D001921:Brain / Q000166:cytology / Q000196:embryology; D002455:Cell Division; D002461:Cell Line, Transformed; D002469:Cell Separation; D005333:Fetus; D005904:Glial Fibrillary Acidic Protein / Q000032:analysis; D005974:Glutamate-Ammonia Ligase / Q000032:analysis; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007381:Intermediate Filament Proteins / Q000032:analysis; D018377:Neurotransmitter Agents / Q000378:metabolism*; D014162:Transfection; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D000952:Antigens, Polyomavirus Transforming; D005904:Glial Fibrillary Acidic Protein; D007381:Intermediate Filament Proteins; D018377:Neurotransmitter Agents; D018698:Glutamic Acid; D005680:gamma-Aminobutyric Acid; D005974:Glutamate-Ammonia Ligase