Mouse ES cells and motor neuron differentiation

ES cells were isolated from mutant mice (WT, Atl1^KI/KI, Reep1^−/−, Atl1^KI/KI/Reep1^−/−). Briefly, E3.5 embryos from naturally mated mice were flushed from oviducts and cultured on primary embryonic mouse fibroblast feeder layers for about a week. ES clones from inner cell mass outgrowths were picked on the basis of morphology and expanded. ES cells were differentiated into motor neurons using a two-step induction protocol (57). ES cells were first primed for neuron induction and embryoid body formation for 2 days with Noggin and fibroblast growth factors. Retinoic acid and smoothened agonist were then added and cultured for 5 days for motor neuron specification. Motor neurons were dissociated and cultured on poly-D/L-ornithine/laminin-coated dishes for an additional 2 weeks when the motor neuron marker choline acetyltransferase (ChAT) was stably expressed.