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. 2023 Sep 14;14:5690. doi: 10.1038/s41467-023-41302-w

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© The Author(s) 2023

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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Fig. 8 — a (i) Current models for pluripotency factor expression; (ii) Pseudotemporal analysis of single-cell RNA-seq trajectories indicates continuous expression of NANOG and OCT4. b Flow cytometry analysis of H9 NANOG-YFP hESCs shows homogeneous YFP expression at D0, D0.5, and D1.5, with a separate YFP-high PGCLC population distinguishable at D2.5 and D3.5. n = 2 biological replicates. Source data are provided as a Source Data file. c Immunostaining of endogenous NANOG, OCT4, and SOX17 protein expression in H9 hESCs from D0 to D3.5 of PGCLC differentiation. Scale bar = 100 μm. Representative images from three independent experiments. d Live imaging analysis of H9 NANOG-YFP hESCs differentiating to PGCLCs at the indicated timepoints Scale bar = 100 μm. Representative images from three independent experiments. e Quantification of PGCLC differentiation efficiencies after NANOG siRNA knockdown at different timepoints during differentiation. Data are presented as mean values ± SEM. n = 4 biological replicates/group for all timepoints except for d3.5 where n = 2 biological replicates. Statistical test: two-way ANOVA with Šídák multiple test correction. Adjusted P values are shown above error bars. Source data are provided as a Source Data file.