(a) Representative traces of spontaneous excitatory postsynaptic currents (sEPSCs; left panels) and corresponding action potential traces elicited by depolarizing current injections (right panels) from the same neuron. Both 250 nM and 1 uM tetrodotoxin (TTX; sodium channel blocker) completely blocked sEPSCs and action potentials. (b) Representative traces of sEPSCs (superimposed 10 sweeps) in the presence or absence of the glutamate receptor antagonists, DNQX (AMPA antagonist) and APV (NMDAR antagonist), and the GABA-A receptor antagonist picrotoxin (PTX). The sEPSCs were blocked by DNQX/APV and PTX further blocked spontaneous activities, suggesting that spontaneous activities observed were result from trans-synaptic transmissions onto the neuron recorded. (c,d) Ten weeks after neuronal differentiation, cells differentiated into NeuN, Hu, or TUJ1-expressing neurons (c) and GFAP or S100β -expressing astrocytes (d). Scale bars, 100 μm. (e-g) Immunolabeling for neuronal markers (MAP2, DCX, or TUJ1) and neurotransmitter phenotype markers, eight weeks after neuronal differentiation, showing that these cells have the potential to differentiate into glutamatergic (glutamate (Glu); e), GABAergic (GABA; f), and glycinergic (GlyT2; g) neurons. Insets show higher magnifications. Scale bars, 50 μm. (h-p) Immunolabeling for neuronal marker DCX and transcription factors that specify neuronal subtypes, including HB9 (h), ISL1/2 (i), LIM1 + 2 (j), BRN3A (k), LBX1 (l), TLX3 (m), CHX10 (n), LHX3 (o), and PAX2 (p)16 two weeks after neuronal differentiation. Scale bars, 40 μm. (q-z) Clonal analysis of H9-derived spinal cord NSCs. Single GFP-expressing H9-derived spinal cord NSC was expanded on GFP negative H9-derived spinal cord NSCs and then purified by a flow cytometry. These subclones differentiated into GFAP-expressing astrocytes (q) and DCX (r) or TUJ1 (s) -expressing neurons. These neurons included HB9 (t) or ISL1/2 (u) -expressing motor neurons and LIM1 + 2-expressing spinal interneurons (v). These interneurons are further characterized and included CHX10 (w) or LHX3 (x) -expressing excitatory interneurons and PAX2 (y) or FOXP2 (z) -expressing inhibitory interneurons. Scale bars, 40 um (q, t-z) and 60 um (r,s). Immunolabeling was independently repeated at least twice with similar results.