Differentiation of Mouse D3 ESCs into Neuronal Precursors

Mouse D3 ESC (ATCC, Manassas, VA, USA) were cultured and induced to form neuronal precursors, resembling the embryonic neuroepithelium that forms the neural tube, as described [28 (link)]. Briefly, undifferentiated (UD) ESC was grown as monolayers, then were induced to form embryoid bodies, then differentiating (D) neuronal precursors were selected from embryoid bodies in attached cultures. D ESC cultures were terminated after four days cultured with or without 0.25 mmol/L buthionine sulfoximine (BSO), a gamma-glutamylcysteine inhibitor [45 (link)], to inhibit the synthesis of glutathione, or 0.001 µmol/L; antimycin A (AA), a mitochondrial complex III inhibitor to stimulate the production of superoxide (both from Sigma-Aldrich, St. Louis, MO, USA).