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. 2020 Jan 27;10:1190. doi: 10.1038/s41598-020-57787-0

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© The Author(s) 2020

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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Confined culture promotes temporal changes in F-actin organization during differentiation. (A) Micropatterned iPSC-derived PF cell colonies that show visibly lower PDX1 intensity (red arrows) are correlated with distinct actin cytoskeleton structures concentrated at the microwell periphery. (B) Characteristic actin structures found throughout 72 hours of differentiation in 150 µm microwells. These structures are grouped based on actin intensity analysis (bottom). The reported intensity profile is obtained from an average of 8 intersecting lines (Fig. S6). (C) Proportion of wells with actin structure (classified in (B) at 24 (n = 10), 48 (n = 19), and 72 (n = 32) hours. Distribution of actin structures changes throughout differentiation and aggregates towards the center in 150 µm microwells. (D) PF colonies in 150 µm microwells with central actin distribution (n = 16) showed increased nuclear PDX1 expression compared to those with peripheral actin distribution (n = 5). Addition of Y-26732 abrogated any increase in nuclear PDX1 expression compared to the unconfined control (n = 12). (E) Addition of Y-26732 ROCK inhibitor 24 hours after seeding prevents actin structural reorganization. *p < 0.05, ****p < 0.001 for the student’s t-test. Scale bars: 100 µm.