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. 2018 Dec 17;9:5339. doi: 10.1038/s41467-018-07740-7

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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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Fig. 3 — rPGCLCs in aggregates correspond to early rPGCs prior to global 5mC erasure. a FACS plots showing the percent of EPCAM/ITGA6 double positive cells in undifferentiated riPSC89 (neg control), and riPSC89 aggregates at Day (D) 1, 2, 4, and 8. b RT-PCR analysis show enrichment of early rPGC markers in rPGCLCs (riPSC89) isolated by FACS at D6 using EPCAM/ITGA6. All data points used to generate the bar graphs (black circles) were overlaid to the corresponding bar graphs, error bars represent the Standard Error of the Mean (S.E.M.). c IF staining of rPGCLCs/rPGCs identified with TFAP2C (red) indicate that the late-stage rPGC marker VASA (green) is not expressed in rPGCLCs at D4 or D8 and instead is expressed in rPGCs in CS12 (D28) and CS23 (D50). Scale bars, 20 µm. d IF staining showing 5mC (green) is present in OCT4-positive(+) (red) rPGCLCs in aggregates (dotted white line) at D2, D4, and D8, while being absent from OCT4⁺ (red) rPGCs (dotted white line) in CS12 (D28) and CS23 (D50) embryos. Scale bars, 20 µm. e IF staining of 5hmC (green) in OCT4⁺ (red) rPGCs (dotted white line) at CS12 (D28) as well as in OCT4⁺ (red) rPGCLCs (dotted white line) in D2, D4, and D8 aggregates. CS23 (D50) rPGCs (OCT4⁺, red) do not have detectable 5hmC (green). Scale bars, 20 µm. Shown are results using riPSC90 unless otherwise stated. N = 3 biological replicates for riPSCs and CS12 (D28); n = 2 biological replicates for CS23 (D50)