Figure 2

Human ESC cells differentiate into glutamatergic neurons.

(A) Schematic of the stem cell neuron differentiation protocol. Days are numbered 0–24 for neuronal differentiation into neural progenitor cells and then renumbered following final plating during neuronal maturation (see methods for details). Solution was replaced every day with the appropriate media plus factors listed above. Vertical, dashed lines indicate cellular plating; the red line indicates final plating into raft arrays or well plates. Representative micrographs of cells during (B) maintenance, (C) differentiation (day 10) and (D, E) maturation (day 1 and 10, respectively). Scale bars, 100 μm. (F) Mature, stem cell-neurons were MAP2-positive and (G) exhibited robust VGLUT1 expression on released rafts. (H) To test for functional activity-dependent synaptic vesicle release in stem cell-neurons, presynaptic terminals were loaded with FM dye (5–95) with KCl and unloaded with electrical stimulation (see methods for details), showing activity-dependent synaptic vesicle release at day 5 on rafts. Scale bar, 10 μm.