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. 2018 Feb 12;8:2814. doi: 10.1038/s41598-018-21098-2

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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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2D Matrigel is preferred to maintain TM morphology and phenotype. Freshly isolated TM cells were cultured on four different substrates and three different media for 4 weeks before subjected to morphological analysis by phase-contrast microscopy (A, scale bar: 100 µm), transcription expression of TM markers (B, C) and ESC and neural crest markers (D,E, using cells cultured on 2D Matrigel in MESCM + 5% FBS as the control, n = 3, *P < 0.05 and ***P < 0.001) and immunostaining of TM markers (F, scale bar: 20 µm).