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. 2022 Feb 15;12:2516. doi: 10.1038/s41598-022-06356-8

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© The Author(s) 2022

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Differentiation of human induced pluripotent stem cells on CPB scaffold into three-germ layers. (A) Representative images of differentiated 253G1 cells at 6–7 days after neuronal ectoderm (top), hepatic endoderm (middle), and cardiac mesoderm (bottom) induction. Left images show the differentiated cells on CPB scaffold induced from the cells maintained on CPB scaffold more than 10 passages before differentiation induction. Right images show differentiated cells on control differentiation substrate according to each protocol from the cells maintained on the control maintenance substrate VTN-N. The control differentiation substrate was Laminin/poly-L-ornithine/poly-L-lysin (iMatrix511/POL/PLL) for neural, Matrigel for hepatic, and laminin (iMatrix-511) for cardiac differentiation. Scale bars are indicated in the images. (B) Expression level of undifferentiated (OCT4 and NANOG) and neural lineage (SOX1, SOX2, PAX6, NCAM1, NES, and NEUROG2) marker genes in the neuro-ectodermal differentiation. (C) Expression level of undifferentiated (OCT4, NANOG, and SOX2) and hepatic lineage (SOX17, CXCR4, GATA4, GATA6m FOXA2, and HNF4A) maker genes in the hepatic endoderm differentiation. (D) Expression level of undifferentiated (OCT4, NANOG, and SOX2) and cardiac lineage (T, NKX2.5, and TNNT2) marker genes in the cardiac mesoderm differentiation. All gene expression levels indicated were examined by RT-qPCR, and indicated as relative expression level to undifferentiated 253G1 cells cultured on VTN-N. Data are presented as mean ± SD of three independent experiments. (*P < 0.05, **P < 0.01 and ***P < 0.001 compared to undifferentiated 253G1 cells cultured on VTN-N).