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. 2014 Jun 5;3(8):888–898. doi: 10.5966/sctm.2013-0213

Figure 5.

Figure 5.

The expression of midbrain DA neuron markers and dopamine release in Atoh1-induced neurons. (A–E): Atoh1-induced pluripotent stem cells were differentiated following the protocol shown in Fig. 4A. DA neurons at differentiation day 36 were immunostained for midbrain DA neuron markers (FOXA2, NURR1, EN1, TH, GIRK2, and DAT) and mature neuron marker (Synapsin). Cell nuclei were counterstained with DAPI. The arrows and arrowhead in (E) indicate DAT+ and DAT− neurons, respectively. Scale bars = 20 μm. (F, G): Representative HPLC chromatogram (F) and quantification (G) of DA and its metabolites (DOPAC, 3-MT, and HVA) released from Atoh1-induced DA neurons at differentiation day 36 in response to KCl-evoked depolarization for 15 minutes. The data represent means ± SEM (n = 2). Abbreviations: DA, dopaminergic; DAPI, 4′,6-diamidino-2-phenylindole; DAT, dopamine transporter; DOPAC, 3,4-dihydroxy-phenylacetic acid; GIRK2, G protein-regulated inward-rectifier potassium channel 2; HVA, homovanillic acid; 3-MT, 3-methoxytyramine; TH, tyrosine hydroxylase.