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. 2018 Nov 7;9:4649. doi: 10.1038/s41467-018-07098-w

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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. 1 — BMI1 inhibits apoptosis and enables chimera formation with hPSCs. a Morphology and DsRed fluorescence expression of UH10-DsRed and UH10-DsRed + BMI1 cell lines. Scale bars, 100 µm. b RT-qPCR analysis of the selected naïve-state pluripotency markers in the indicated cells. Naïve-state hPSCs were converted according to the protocol from Austin Smith’s lab (3i, Methods). Error bars represent mean + SEM of three independent replicates. **p < 0.01, ***p < 0.001. c Alkaline phosphatase staining on colonies formed by the indicated cells after plating at 2000 single cells per well of 6-well-plate for 7 days. d, e Merged images and statistics of the numbers of DsRed⁺ cells in injected mouse blastocysts after injection of the ten indicated DsRed⁺ cells in the later morulas or early blastocysts and 1-day culture in vitro; scale bars, 50 µm, mean ± SEM of 21 (UH10-DsRed) or 14 (UH10-DsRed + BMI1) samples, ***p < 0.001. f Representative fluorescence images of mouse embryos stained with Annexin V after injection of ten indicated DsRed⁺ cells in the later morulas or early blastocysts and 1-day culture in vitro. White arrow, DsRed⁺/Annexin V⁺ cells; Yellow arrow, DsRed⁺/Annexin V⁻ cells. Scale bars, 20 µm. g Statistics of the percentage of engrafted DsRed⁺ cells with Annexin V⁺ in the mouse blastocysts; mean + SEM of 21 (UH10-DsRed) or 14 (UH10-DsRed + BMI1) samples, ***p < 0.001. h E10.5 mouse embryos derived from UH10-DsRed + BMI1 in bright field and fluorescence. Scale bars, 1 mm. i Representative quantitative genomic PCR analysis of the human mitochondria DNA in E10.5 mouse embryos after injection of the indicated cells. A human DNA control (H), a mouse DNA control (M) and a series of human−mouse cell dilutions (1/100 to 1/1000,000) were run in parallel to estimate the degree of human cell integration. The dashed line indicates the detection level of one human cell in 10,000 mouse cells. j Summary of chimera assays of the indicated DsRed⁺ cells injected at the later morula or early blastocyst stage. Dox were added in culture or fed to recipient for BMI1 overexpression until analysis, otherwise indicated