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. 2017 Nov 22;7:16001. doi: 10.1038/s41598-017-16248-x

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© The Author(s) 2017

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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Ground state naïve mESC, compared to serum/LIF-dependent mESC, improve neural specification. S/L-dependent and naïve mESC, labeled with (Ai,Bi) DAPI, show indistinguishable and high expression of pluripotency transcription factors (Aii,Bii) Oct4 and, (Aiii,Biii) Sox2 prior to differentiation. (Aiv,Biv) Merge images. (C) Transcriptional profiling confirmed expression of pluripotency genes in undifferentiated naïve and S/L-dependent mESCs. Data represents change in gene expression levels in S/L-dependent mESC (bars), compared to naïve cultures (normalized to 1, dotted line). (D) Reflective of differences in pluripotency states, S/L-dependent mESCs showed elevated Wnt7a and Id1, as well as downregulation of Bmp7 expression, compared to naïve mESC. (E) Schematic depicting the effect of LIF and/or 2i initiated neural differentiation from S/L-dependent verses naïve mESC. (E”) Transient exposure of naïve PSC to LIF resulted in improved neural specification, and elimination of PSC upon differentiation. (F) Flow cytometry plots show high viability of differentiated S/L-dependent mESC at day7, (Fi) yet poor survival of naïve mESC differentiation from 2i media, (Eii) an effect that could be circumvented by transient culturing in LIF media. Flow cytometry plots for (G) Oct4, (I) Nestin, (K) Pax6 and (M) FoxA2 in undifferentiated and day 7 differentiated cultures. Comparative quantitative analysis of FACS plots for S/L-dependent mESC and naïve mESCs at day7 after differentiation for (H) Oct4, (J) Nestin, (L) Pax6 and (N) FoxA2. Upon differentiation, naïve ESC cultures showed reduced proportions of contaminating Oct4+ PSC, elevated neuralization (Nestin+) and appropriate default dorsal forebrain specification, as indicated by reduced FoxA2+ and elevated Pax6+ cells in culture. Representative images of (O,S) Oct4, (P,T) Nestin, (Q,U) Pax6 and (R,V) FoxA2 immunostaining from primed and naïve mESCs differentiated under neural conditions for 7days. Note the absence of both Oct4+ PSCs and FoxA2 off-target ventral neural progenitors, as well as expansive Nestin+ and Pax6+ NPC in cultures derived from naïve mESC, compared to S/L-dependent mESC. Data (n = 4) represents mean ± SEM, Students t-test. Scale bars: A-B = 100 um, O-V = 50um. *p < 0.05, **p < 0.01, ***p < 0.001. mESC: mouse embryonic stem cell; NPC: Neural pluripotent stem cells; PSC: pluripotent stem cells; S/L: Serum and LIF.