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. 2019 Jul 5;9:9763. doi: 10.1038/s41598-019-45100-7

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© The Author(s) 2019

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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Forced Expression of Pax6 Restores Neural Crest Progenitor Status in P10 LNC. P10 LNC on coated Matrigel in MESCM was transfected with Ad-GFP (GFP) or Ad-GFP-Pax6 (GFP-Pax6) plasmid at MOI 100 for 4 days, then the medium was switched to NSCM neurosphere medium for 7 days. Neurospheres were imaged by confocal microscopy with or without fluorescence for GFP (A). The total numbers of neurospheres with a size greater than 50 μm in diameter were compared (B, *p = 0.001, n = 3). The differentiation potential for cells derived from neurospheres was assessed after cells were cultured in different induction media and observed by phase microscopy and immunofluorescence staining of neurofilament M (NFM, red), O₄ (red), and glial fibrillary acidic protein (GFAP, green) (C, nuclear counterstaining by Hoechst 33342, scale bars = 50 µm).