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. 2020 Sep 16;10:15209. doi: 10.1038/s41598-020-72214-0

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© The Author(s) 2020

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Expression of selected neuronal markers in hBORGs. (A) Representative images of comparison of neuronal and glial markers expression by co-immunostaining in hBORGs sections in panel a. At day 14 after differentiation, βIII-Tubulin Tuj1 + neurons (panel b) and GFAP + astrocytes (panel c) were identified without a preferential localization. At day 14, few cells were Nestin + /SOX2 + (panel d and e). Scale bar in panel a and d is 100 μm, in panel b and c is 50 μm and in panel e is 10 μm. (B) Representative images of neuronal lineage markers. hBORGs sections from day 14 showed that most of differentiated neurons expressed VGlut1 (panel a and b), marker of glutamatergic neuronal lineage. Minimal expression of VGAT, marker of GABAergic neuronal lineage, was observed in hBORGs sections from day 28 and was maintained up to day 180 (panels c, d and e), whereas the expression of tyrosine hydroxylase (TH), marker of dopaminergic neuronal lineage, is evident only at day 180 (panels e and f). Scale bar in panels a, c, d and e is 100 μm and in panels b is 10 μm and f is 50 μm. (C) Expression of synaptic markers synaptophysin (SYN), PSD95 were observed as early as day 14 of differentiation (panel a-c) (D) Cell viability in hBORGs was assessed by live/dead assay followed by confocal microscopy. Images represent live cells (green) and dead cells (red) from one of the representative experiments (N = 3). Scale bar is 100 μm.