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. 2018 Apr 12;8:5907. doi: 10.1038/s41598-018-24074-y

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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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Optimization of mesoderm induction for cardiac differentiation from RhiPSCs. (a) Immunofluorescence staining for pluripotency marker OCT4 and early mesoderm marker Brachyury (BRA) on RhiPSC cultured in different media after EDTA dissociation. Nuclei were stained by DAPI. Scale bars, 100 µm. (b) Immunofluorescence staining for OCT4 and Brachyury (BRA) during the first three days’ differentiation to access the dynamic profiles of losing pluripotency and gaining mesoderm identities under different mesoderm induction treatments. Nuclei were stained by DAPI. Scale bars, 100 µm. (c) Real-time RT-PCR showed daily expression profiles of cardiac progenitor markers (MESP1, PDGFRα, KDR, and MEF2c) and neural progenitor markers (NESTIN and PAX6) under different differentiation conditions (data are mean ± SEM, n = 3). (d) Representative flow cytometry analysis from more than 5 times experiments of cTnT expression under CHIR, BA or BAF treatment as shown in (B). (e) Screening of different mesoderm induction conditions in RhiPSC cardiac differentiation. Flow cytometry results of cTnT antibody at 10 days after differentiation were shown. The number after letter F was the concentration (ng/ml) of FGF2. CM, conditioned medium; KSR, knockout serum replacement; CHIR, CHIR99021; BA, BMP4 + Activin A; BAF, BMP4 + Activin A + FGF2.