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. 2018 Sep 14;8:13804. doi: 10.1038/s41598-018-31772-0

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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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hiPSC characterization, quantification of pluripotency and differentiation markers. (a) Example of hiPSC WT and R219H colonies (x100). (b) Relative gene expression of pluripotency markers (OCT4, NANOG and REX1) assessed by qRT–PCR. The gene expression was normalized to the expression measured in human embryonic stem cells reference standards (HES2 cells). (c) Flow cytometry quantification of cells expressing the pluripotency–associated proteins (SSEA4, Tra–1–60, OCT4). (d) After embryoid body formation, the induction of germ lineage–specific markers was evaluated by qRT–PCR and compared with the same non–differentiated hiPSCs. The AFP, HAND1 and PAX6 genes were used as markers of endoderm, mesoderm and ectoderm. (e) The karyotype of hiPSC was evaluated by G–banding analysis. Both WT (left) and R219H (right) demonstrated normal karyotype. (f) The presence of the R219H point mutation in Na_v1.5 was confirmed in genomic DNA of hiPSC cells from the patient while the mutation is absent in hiPSC from the healthy control father.