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. 2015 May 21;5:9758. doi: 10.1038/srep09758

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Copyright © 2015, Macmillan Publishers Limited

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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Validation of GEPA predicted regulatory pathways using in vitro cardiovascular differentiation from human iPS cells. (a-d) Validating the roles of well-known signaling pathways in human cardiovascular differentiation. (a) A scheme of modulating BMP and Wnt pathways from day1 to day4. No factor was added from day 0 to day 1 and ActivinA (3 ng/ml) was added into all conditions through day 1 to day 4. (b) Representative images showing EBs of day 20. Scale bar, 100 μm. (c) Quantitative analysis of EB numbers at day 20. Statistical analysis were performed with unpaired t-test, *p < 0.01 (d) Quantitative analysis of ratio of CM at day 20 EBs. EBs were dissociated, immunostained with anti-CTNT antibody and FACS analyzed. CTNT+ cells represent CMs. Statistical analysis were performed with unpaired t test, *p < 0.1 (e) A scheme of inhibiting ephrin receptor signaling pathway using lithocholic acid (LA), which is a small molecule inhibitor of ephrin receptors, from day 4 to day 20 of differentiation in human iPS cells. DMSO treatment served as control. (f) Representative images showing day 20 EBs in DMSO and LA treated wells. Scale bar, 100 μm. (g) FACS analysis of ratios of CTNT+ CMs from day 20 EBs. Results were represented as mean ± S.D., from 3 independent experiments, statistical analysis were performed with unpaired t test, *p < 0.01.