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. 2018 Jan 16;8:804. doi: 10.1038/s41598-018-19291-4

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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mDA differentiation protocol yields mDA NPCs at day 14 and mDA neurons at day 42. (A) mDA differentiation scheme. After dissociation, iPS cells were kept in suspension culture for 21 days. In the first 14 days, cells were induced with DM (Dorsomorphin), SB431542, SAg 1.3 (Smoothened agonist), Pur (Purmorphamine), and CHIR99021. From day 14 through day 21, cells were differentiated in the neuronal differentiation medium containing BDNF, GDNF, dbcAMP, Ascorbic acid, and DAPT. From day 21, cells were further differentiated in the terminal differentiation medium containing BDNF, GDNF, dbcAMP, Ascorbic acid, and Ara-C. (B) Immunostaining of day 14 (top two rows) and day 42 (bottom two rows) 18a cells. (C) The mean concentration (pg/ml) of dopamine released by day 0 cells and day 42 18a cells. (D) Phase contrast image showing human iPSC 18a-derived dopaminergic neuron cultures after 1 month adherent culture. Arrowhead points to a recorded cell. (E) Representative traces showing whole-cell voltage-gated Na⁺ and K⁺ currents recorded in human iPSC 18a-derived dopaminergic neuron culture. (F) Representative traces showing responses to GABA and AMPA (100 representative traces each) (G) Representative traces showing spontaneous action potentials. The resting membrane potential was −50 mV.