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. 2019 Jan 29;9:913. doi: 10.1038/s41598-018-37485-8

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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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Sequential transfection of a cocktail of TFs along with EMX2, stimulating factor of mesenchymal-to-epithelial transition, is essential for epithelialization. (a) Diagram of differentiation. (b) Schematic of the tentative differentiation protocol from hESCs. (c) Relative mRNA expression levels of SNAI2 and VIMENTIN, mesenchymal markers, on day 4 in cells differentiated from hPSCs using protocol depicted in; 4TFs, syn-FIGLA, syn-PITX2, syn-ASCL1, and syn-TFAP2C; 3TFs, syn-HNF1A, syn-GATA3, and syn-GATA1. (d) Immunocytochemistry for CDH1, epithelial marker, on day 4 in cells differentiated from hPSCs after transfecting 4TFs + 3TFs and 4TFs + 3TFs + EMX2; 4TFs, syn-FIGLA, syn-PITX2, syn-ASCL1, and syn-TFAP2C; 3TFs, syn-HNF1A, syn-GATA3, and syn-GATA1. Scale bar, 100 μm. (e) Relative mRNA expression levels of PAX8 and LHX1, markers of pretubular aggregate, on day 4 in cells differentiated from hPSCs after transfecting 4TFs + 3TFs and 4TFs + 3TFs + EMX2; 4TFs, syn-FIGLA, syn-PITX2, syn-ASCL1, and syn-TFAP2C; 3TFs, syn-HNF1A, syn-GATA3, and syn-GATA1. Values shown are the means ± SEM. P-values were determined by a Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001.