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. 2017 Oct 20;7:13671. doi: 10.1038/s41598-017-13826-x

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© The Author(s) 2017

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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Cellular populations after ATRA-mediated differentiation of EC cells. (A) Experimental design of embryoid body differentiation, retinoid treatments, and sample collection. (B) Neurons, astrocytes and microglial cells were assessed in ATRA-differentiated EC cells by immunofluorescent detection of TUJ1, GFAP and IBA1, respectively (see left panels for an example, and histograms for quantification). (C) Immunofluorescence analyses of excitatory glutamatergic (GluR2/3⁺) and inhibitory (GAD65/67⁺) neurons. (D) Immunofluorescent detection of DARPP32⁺ medium-spiny neurons (MSNs) within neuronal (Neurofilament, NF⁺) cells. (E) mRNA expression levels of striatonigral and striatopallidal MSN markers quantified by qPCR. Expression is plotted as fold change as compare to expression level in undifferentiated cells. *p < 0.05; **p < 0.01; ***p < 0.001 as compare to undifferentiated cells. (F) Induction of c-Fos expression measured by RT-qPCR, 90 min after haloperidol (10 µM), dopamine (1 µM) or SKF81297 (1 µM) treatment. Expression is plotted as a fold change with respect to control cells treated with vehicle. ***p < 0.001 as compared to vehicle treatment. Throughout the figure, graph bars represent mean ± s.e.m. for 3 independent experiments for immunocytochemistry and 5 independent experiments for qPCR. n.d., not detected.