Electrical and neurotransmitter activity of mature neurons derived from mouse embryonic stem cells by Sox-1 lineage selection and directed differentiation.
Sx1TV2/16C is a mouse embryonic stem (ES) cell line in which one copy of the Sox1 gene, an early neuroectodermal marker, has been targeted with a neomycin (G418) selection cassette. A combination of directed differentiation with retinoic acid and G418 selection results in an enriched neural stem cell population that can be further differentiated into neurons. After 6-7 days post-plating (D6-7PP) most neurons readily fired tetrodotoxin (TTX)-sensitive action potentials due to the expression of TTX-sensitive Na(+) and tetraethylammonium (TEA)-sensitive K(+) channels. Neurons reached their maximal cell capacitance after D6-7PP; however, ion channel expression continued until at least D21PP. The percentage of cells receiving spontaneous synaptic currents (s.s.c.) increased with days in culture until 100% of cells received a synaptic input by D20PP. Spontaneous synaptic currents were reduced in amplitude and frequency by TTX, or upon exposure to a Ca(2+)-free, 2.5 mm Mg(2+) saline. S.s.c. of rapid decay time constants were preferentially blocked by the nonNMDA glutamatergic receptor antagonists CNQX or NBQX. Ca(2+) levels within ES cell-derived neurons increased in response to glutamate receptor agonists l-glutamate, AMPA, N-methyl-d-aspartate (NMDA) and kainic acid and to acetylcholine, ATP and dopamine. ES cell-derived neurons also generated cationic and Cl(-)-selective currents in response to NMDA and glycine or GABA, respectively. It was concluded that ES-derived neurons fire action potentials, receive excitatory and inhibitory synaptic input and respond to various neurotransmitters in a manner akin to primary central neurons.
Lang
R J
RJ
Department of Physiology, Monash University, Clayton, Victoria 3800, Australia. rick.lang@med.monash.edu.au
Haynes
J M
JM
Kelly
J
J
Johnson
J
J
Greenhalgh
J
J
O'brien
C
C
Mulholland
E M
EM
Baker
L
L
Munsie
M
M
Pouton
C W
CW
D018750:6-Cyano-7-nitroquinoxaline-2,3-dione / Q000494:pharmacology; D000200:Action Potentials / Q000187:drug effects / Q000502:physiology*; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian; D018698:Glutamic Acid / Q000494:pharmacology; D006609:High Mobility Group Proteins / Q000502:physiology*; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D018377:Neurotransmitter Agents / Q000494:pharmacology*; D055748:SOXB1 Transcription Factors; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology
D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D018377:Neurotransmitter Agents; D055748:SOXB1 Transcription Factors; C098693:Sox1 protein, mouse; D018698:Glutamic Acid; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione