Abstract
We investigated apoptosis during early stages of in vitro differentiation of neuronal precursors generated by embryonic day 14 (E14) mouse striata stem cells. Differentiation was in conditions of suboptimal growth factor supply. Apoptosis reached 10-15% of cells and affected proliferating as well as postmitotic cells, including TUJ1-positive cells. Inhibition of apoptosis led to an increased proportion of TUJ1-positive cells generated by stem cells. K(+) current was reported to be related to apoptosis. Outward K(+) currents were present in differentiating neuronal precursors that were consistent with delayed rectifier and transient A-type currents. The amplitude of the delayed rectifier current varied during the first 4 days of stem cell differentiation. Current amplitude was greatly increased in the presence of staurosporine but reduced at elevated extracellular K(+) concentration. In addition, the amplitude of the current was significantly diminished by inhibiting several caspases, but not caspase 8. In Bax knock-out transgenic neuronal precursors, K(+) current was not decreased after the first day but at later stages of cell differentiation. At this early stage, apoptosis of proliferating cells and of TUJ1-positive cells was not reduced by the absence of Bax, but was by caspase 9 inhibition. Thus, activation of a delayed rectifier K(+) current in differentiating stem cells is related to apoptosis. Recordings of this current revealed that apoptosis at early stages of neuronal differentiation occurred in two phases that did not exhibit similar dependence on the proapoptotic protein Bax and that probably used different pathways.
Publication types
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Comparative Study
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Chloromethyl Ketones / pharmacology
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Animals
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Annexin A5 / metabolism
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Apoptosis / genetics
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Apoptosis / physiology*
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Barium / pharmacology
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Biomarkers
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Caspase Inhibitors
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Caspases / metabolism
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Caspases / pharmacology
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Cell Count / methods
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Cell Differentiation / drug effects
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Cell Differentiation / genetics
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Cell Differentiation / physiology*
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Cells, Cultured
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Embryo, Mammalian
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Enzyme Inhibitors / pharmacology
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Glial Fibrillary Acidic Protein / metabolism
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Immunohistochemistry / methods
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Intermediate Filament Proteins / metabolism
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Ion Channel Gating / genetics
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Ion Channel Gating / physiology
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Maximum Tolerated Dose
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Membrane Potentials / drug effects
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Membrane Potentials / physiology
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Mice
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Mice, Knockout
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Nerve Tissue Proteins / metabolism
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Nestin
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Neurons / drug effects
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Neurons / physiology*
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Patch-Clamp Techniques / methods
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Phosphopyruvate Hydratase / metabolism
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Potassium Channel Blockers / pharmacology
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Potassium Channels, Voltage-Gated / genetics
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Potassium Channels, Voltage-Gated / physiology*
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Potassium Chloride / pharmacology
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Proliferating Cell Nuclear Antigen / metabolism
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Proto-Oncogene Proteins / deficiency
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Proto-Oncogene Proteins / genetics
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Proto-Oncogene Proteins c-bcl-2*
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Staurosporine / pharmacology
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Stem Cells / drug effects
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Stem Cells / physiology*
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Tetraethylammonium / pharmacology
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Time Factors
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Tubulin / metabolism
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bcl-2-Associated X Protein
Substances
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Amino Acid Chloromethyl Ketones
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Annexin A5
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Bax protein, mouse
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Biomarkers
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Caspase Inhibitors
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Enzyme Inhibitors
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Glial Fibrillary Acidic Protein
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Intermediate Filament Proteins
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Nerve Tissue Proteins
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Nes protein, mouse
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Nestin
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Potassium Channel Blockers
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Potassium Channels, Voltage-Gated
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Proliferating Cell Nuclear Antigen
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Proto-Oncogene Proteins
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Proto-Oncogene Proteins c-bcl-2
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Tubulin
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bcl-2-Associated X Protein
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benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone
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beta3 tubulin, mouse
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Barium
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Tetraethylammonium
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Potassium Chloride
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Caspases
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Phosphopyruvate Hydratase
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Staurosporine