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. 2017 Nov 22;7:16001. doi: 10.1038/s41598-017-16248-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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Naïve mESCs differentiated to a caudal ventral forebrain fate, show appropriate regional and temporal specification. (A) Neural differentiation protocol, detailing morphogen and small molecules employed, to generate ventral forebrain progenitors and neurons from naïve PSC. (B–E) Flow cytometry plots at day 7 and, (F) quantitative analysis at 0 and 7 for Oct4, Nestin, Pax6 and FoxA2, showing appropriate downregulaton of pluripotent (Oct4) and dorsal (Pax6) markers together with upregulation of neural (Nestin) and ventral (FoxA2) protein expression. (G) Transcriptional downregulation of Pax6 together with the upregulation of Nhlh2 and OTP suggested these cultures included neural progenitors of a ventral hypothalamic fate. (H) Representative photomicrographs depicting expression of Nestin, Otx2 and FoxA2 in ventral forebrain progenitors at day7. (I) Maturation of cultures resulted in the generation of GABA+ TUJ+ neurons, as well as FoxA2+ and TH+ neurons (showing low co-localisation), suggestive of hypothalamic neurons. Data (n = 3) represents mean ± SEM, Students t-test. Scale bar = 100 um. *p < 0.05, ***p < 0.001.