Differentiation of murine ES cells into motor neurons. The scheme outlines the process of motor neuron differentiation from ES cells (A). Murine ES cells (HBG3) were expanded on a layer of primary mouse embryonic fibroblasts. Undifferentiated stem cells form round clusters (arrows) as they proliferate on top of a feeding fibroblast layer, and they have weak GFP expression (B-B'). Stem cells were separated from the fibroblast layer and plated in low attachment dishes to allow for differentiation. Two-day-old stem cell aggregates (called embryoid bodies, EB, arrows) were induced to differentiate with 1 μM retinoic acid and 1 μM sonic hedgehog agonist SAG for 5 days. Differentiated ES cells have strong GFP expression (C-C'). When plated on poly-DL-ornithine/laminin/matrigel coated plates, the differentiated ES cells extend long neurites after 2 days in culture (D-D'). Representative phase contrast images for 1B-1D are shown on the top and corresponding fluorescent images are on the bottom. Scale bar = 200 μm except as indicated. MN = motor neuron.