GABAergic lineage differentiation of AF5 neural progenitor cells in vitro.
We have previously described an immortal rat central-nervous-system progenitor cell line, AF5, which is able to exit the cell cycle and assume a differentiated state with neuronal properties. The phenotypic specification of differentiated AF5 cells, however, is not known. In the present study, when induced to differentiate by serum starvation in Neurobasal medium, AF5 cells down-regulate glial fibrillary acidic protein and up-regulate expression of beta-III-tubulin, medium-molecular-weight neurofilament protein, and neuronal growth-associated protein 43. Expression of the gamma-aminobutyric acid (GABA) lineage marker, glutamic acid decarboxylase 67 (GAD67), increases during differentiation, suggesting that AF5 cells adopt a GABAergic lineage. Time-course analysis of the GABAergic neuron specification transcription factor, Pitx2, by reverse transcription/polymerase chain reaction, has shown an increase in the Pitx2 transcript 48 h after initiation of differentiation. In differentiated AF5 cells, expression of the Pitx2 target gene products GAD65 and GABA transporter-1 increases. Cellular GABA levels in differentiated AF5 cells increase by about 26-fold, and GABA release into the medium is 150-fold higher compared with that of undifferentiated cells. Therefore, AF5 cells can be induced to differentiate to a neuronal phenotype with a GABAergic lineage.
Sanchez
Joseph F
JF
Development and Plasticity Section, Cellular Neurobiology Research Branch, Intramural Research Program, National Institute on Drug Abuse, NIH, DHHS, Baltimore, MD 21224, USA. jsanchez@mail.nih.gov
Crooks
Daniel R
DR
Lee
Chun-Ting
CT
Schoen
Cynthia J
CJ
Amable
Rose
R
Zeng
Xianmin
X
Florival-Victor
Thierry
T
Morales
Nelly
N
Truckenmiller
Mary E
ME
Smith
Donald R
DR
Freed
William J
WJ
D000818:Animals; D002454:Cell Differentiation; D019070:Cell Lineage; D002478:Cells, Cultured; D003470:Culture Media / Q000494:pharmacology; D005786:Gene Expression Regulation; D005968:Glutamate Decarboxylase / Q000378:metabolism; D018398:Homeodomain Proteins / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D009474:Neurons / Q000166:cytology*; D051381:Rats; D013234:Stem Cells / Q000166:cytology*; D014157:Transcription Factors / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000096:biosynthesis / Q000378:metabolism*; D000097577:Homeobox Protein PITX2
D003470:Culture Media; D005968:Glutamate Decarboxylase; D018398:Homeodomain Proteins; D007527:Isoenzymes; D014157:Transcription Factors; D005680:gamma-Aminobutyric Acid; D000097577:Homeobox Protein PITX2; C402565:glutamate decarboxylase 1