AF5, a CNS cell line immortalized with an N-terminal fragment of SV40 large T: growth, differentiation, genetic stability, and gene expression.
Central nervous system progenitor cells that are self-renewing in culture and also differentiate under controlled conditions are potentially useful for developmental studies and for cell-based therapies. We characterized growth and plasticity properties and gene expression in a rat mesencephalic cell line, AF5, that was immortalized with an N-terminal fragment of SV40 large T (T155g). For over 150 population doublings in culture, the growth rate of AF5 cells remained steady, the cells remained responsive to bFGF, and telomerase activity and telomere lengths were unchanged. While karyotype analyses revealed some chromosomal abnormalities, these were also unchanged over time; additionally, no mutations in p53 gene sequences were found, and wild-type p53 activation was normal. AF5 cells produced PDGF, TGFbeta1, TGFbeta2, and bFGF. Similar to primary progenitor cells, AF5 cells retained their plasticity in culture; they could be propagated in an undifferentiated state as "neurospheres" in serum-free media or as adherent cultures in serum-containing media, and they differentiated when allowed to become confluent. Adherent subconfluent actively growing cultures expressed a marker for immature neurons, nestin, while few cells expressed the mature neuronal cell marker betaIII-tubulin. Confluent cultures ceased growing, developed differentiated morphologies, contained few or no nestin-expressing cells, and acquired betaIII-tubulin expression. Global gene expression was examined using a 15,000 gene microarray, comparing exponential growth with and without bFGF stimulation, and the differentiated state. The AF5 cell line exhibited stable genetic and growth properties over extended periods of time, while retaining the ability to differentiate in vitro. These data suggest that the AF5 cell line may be useful as an in vitro model system for studies of neural differentiation.
Truckenmiller
M E
ME
Cellular Neurobiology Research Branch, National Institute on Drug Abuse, 5500 Nathan Shock Drive, Baltimore, Maryland 21224, USA.
Vawter
Marquis P
MP
Zhang
Peisu
P
Conejero-Goldberg
Concha
C
Dillon-Carter
Ora
O
Morales
Nelly
N
Cheadle
Chris
C
Becker
Kevin G
KG
Freed
William J
WJ
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry / Q000235:genetics*; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002461:Cell Line, Transformed / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D007621:Karyotyping; D008636:Mesencephalon / Q000166:cytology*; D020411:Oligonucleotide Array Sequence Analysis; D010446:Peptide Fragments / Q000235:genetics; D010641:Phenotype; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism / Q000502:physiology; D019098:Telomerase / Q000378:metabolism; D016615:Telomere / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism; D053773:Transforming Growth Factor beta1; D053781:Transforming Growth Factor beta2; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
D000952:Antigens, Polyomavirus Transforming; D010446:Peptide Fragments; D010982:Platelet-Derived Growth Factor; C508962:Tgfb1 protein, rat; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D053781:Transforming Growth Factor beta2; D016159:Tumor Suppressor Protein p53; D016222:Fibroblast Growth Factor 2; D019098:Telomerase