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. 2017 Apr 10;7:766. doi: 10.1038/s41598-017-00774-9

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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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Aggregation of stem cells under hypoxia. (a) Pluripotency of IMR90.4 iPSCs confirmed by expression of SOX2, OCT4, NANOG and SSEA4. (b) Forced aggregates at day 5 (D5) grown in KSR based neural induction medium (NIM) or BE6.2 medium based NIM in 20% O₂. Arrow in the BE6.2 sample indicates early vesicle formation. (c) Diagram illustrating the approach used to study 1-day recovery and aggregation of dissociated PSCs in 5 or 20% O₂ followed by 20% O₂. (d) A time-lapse sequence of images taken after 24 hours and from D6-10 illustrating initiation and elaboration of individual vesicles (see dark arrows). In yellow is a typical region selected for manual excision. (e) Forced aggregates (n = 15 per time point) in 5% and 20% O₂ imaged at daily increments, measured along their radial diameter and quantitated. Average diameters plotted as a function of days in culture and statistical significance determined using the student t-test with Holm-Sidak method. *Statistically significant (p < 0.05), n.s. = not significant. Scale bars (a) = 200 μm; (b,d) = 300 μm.