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. 2017 Nov 28;8:1825. doi: 10.1038/s41467-017-01946-x

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© The Author(s) 2017

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 Commonslicense, unless indicated otherwise in a credit line to the material. If material is not included in the article’sCreative 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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Fig. 4 — Increased cell maturation and size in low-density cardiopatches. a Relative expression levels of 10 maturation genes in d0, 1 week, 2 week, and 3 week cardiopatches compared with those in adult human left ventricles (LV). Linear regression from d0 to 3 weeks: TNNI3, MYL2, MYOM2, CASQ2, p < 0.0001; MYOM3, CKMT2, p < 0.014; S100A1, COX6A2, CKM, p < 0.006; PLN, p = 0.1081. *p < 0.05, **p < 0.001, ***p < 0.0001 vs. d0; ^# p < 0.023, ^## p < 0.0006, ^### p < 0.0001 vs. 1 week; ^† p < 0.037, ^†† p < 0.0037 vs. 2 week, via post hoc Tukey’s tests. For clarity, only 3-week statistical comparisons are shown. b Relative gene expression in 0.5MM vs. 1MM cardiopatches; *p < 0.041, **p < 0.0073, ***p < 0.0001, post hoc Tukey’s tests. c Protein/DNA ratio in 0.5MM vs. 1MM cardiopatches; n = 3/4 patches (1/0.5MM). d Representative western blots for myosin heavy chain-β (βMHC), SAA, lamin B1 (LamB1), GAPDH, and Cx43 in d0 cells and 3-week 0.5MM and 1MM cardiopatches; dotted line indicates lanes spliced from the same gel. e Quantified protein levels in d0 cells, 1MM and 0.5MM cardiopatches normalized to nuclear envelope protein LamB1, shown relative to 1MM cardiopatches; **p < 0.01, ***p < 0.001 vs. d0; ^† p < 0.05, ^†† p < 0.01, ^††† p < 0.001 vs. 1MM, post hoc Tukey’s tests. For gene expression studies (a, b), n = 6 patches from two differentiations; for protein studies (c–e), n = 8/10 patches (1/0.5MM) from three to four differentiations. f Representative low-magnification view of cell nucleus (nuc) and surrounding sacromeric structures (sarc) in 3-week-old 0.5MM cardiopatches. Scale bar 2 µm. g Localization of I-bands, Z-discs, and H-zones within hiPSC-CM sarcomeres. Scale bar 1 µm. h Average number of H-zones and I-bands per sarcomere; n = 4 patches from two differentiations, data compiled from a total of 74 random fields of view. i Mitochondria (mito) were found positioned alongside CM myofibrils. Scale bar 500 nm. j, k Evidence of M-bands (j) and T-tubular-like structures (T-t, k) in 3-week-old 0.5MM cardiopatches. Scale bars 500 nm. All data are presented as mean ± SEM