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. 2019 Nov 5;9:16006. doi: 10.1038/s41598-019-52516-8

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© The Author(s) 2019

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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Characterization of CMCs after passages under adherent culture conditions in ABC medium. (a) Morphology of cultured CMCs derived from RH5, RH6, and iPS lines in ABC medium. (b) Immunofluorescence analysis of MESP1 and Ki67 expressions in CMCs cultured in ABC medium. Cells were counter-stained with DAPI. (c) Representative graphs show the percentages of MESP1⁺ and Ki67⁺ cells in cultured CMCs derived from RH5, RH6, and iPS lines at early (1–2) and late (9–10) passages as analyzed by flow cytometry. (d) PDGFRα and CD56 surface marker analysis of CMCs at the late passages. (e) Karyotype image of cultured RH5-derived CMCs in ABC medium at passage 10 (P10) that shows the normal cell karyotype. (f) Representative graph of total cell counts generated after nine passages of RH5-, RH6- and iPS-derived CMCs in ABC medium. (g,h) Doubling time and expansion fold of CMCs at early and late passages. Scale bars: 200 µm. All data: mean ± SD. The statistical differences between P4 and P10 in all experimental groups were analyzed by unpaired t-test.