Figure 2

Reducing hiCM density by sparse plating increases proliferative capacity. (A) YAP and β-catenin in dense and sparse hiCMs using 20 × widefield fluorescence microscopy. (B) Quantification of nuclear/total YAP ratio. Significance determined by paired two-tailed Student’s t test. (N = 23 dense cells and 27 sparse cells from 3 independent experiments) (C) Ki67 and β-catenin immunolocalization in dense and sparse hiCMs using 20 × widefield fluorescence microscopy. (D) Percent of Ki67-positive nuclei in dense (N = 8046 hiCMs from 4 experiments) and sparse (N = 1716 cells from 3 experiments) hiCMs. Significance determined by an unpaired two-tailed Student’s t test. (E) Fucci cell cycle probe transduced into dense or sparse hiCMs. Arrows shows S–M phase. (F) Proliferation of dense or sparse hiCMs quantified by counting nuclei fold change over 48 h (N = 281,250 cells from 5 independent experiments). Significance determined by unpaired two-tailed Student’s t test. (G) RNASeq Volcano plot of genes significantly changed in sparse versus dense hiCMs (N = 300,000 hiCMs per treatment from 3 independent experiments). A negative fold change represents a downregulation in sparse hiCMs. A positive fold change represents an upregulation in sparse hiCMs. Gene names of determined YAP target genes (including CTGF and CYR61), cardiac myocyte identity genes (TNNT2 and ACTN2), and cardiac myocyte maturity genes (including GATA4 and FOXOD) can be found in Tables 1, 2, 3.