Fig. 3.

Culture in MM induces transcriptional and proteomic maturation in hCOs. (A) PCA of RNA-seq data using a subset of genes identified by Delaughter et al. (27) to profile maturity (genes from table S6 in ref. 27). RNA-seq data derived from hCOs cultured in CTRL medium or MM (n = 4 experiments, each 20 pooled hCOs) and our human adult heart sample (n = 1, three pooled hearts) combined with RNA-seq data from Gene Expression Omnibus accession number GSE62913 (25) containing 2D hPSC-CMs at 20 d (n = 3) and 1 y (n = 3), human fetal ventricles (n = 2), and human adult hearts (n = 2). All genes were >10 counts per million for at least one sample. Bar at the top is a projection of PC1, the component that correlates with maturation. (B) Pearson correlation coefficient indicates that hCOs cultured in MM are significantly correlated to human adult heart tissue. Graph is of log₂(cpm) of human adult heart (n = 1, three pooled hearts) or average gene expression of hCOs cultured in MM (n = 4). (C) Pearson correlation coefficient indicates that hCOs cultured in MM are significantly correlated to human adult heart tissue. Graph is of log₂(iBAQ) of human adult heart (n = 1) or pooled hCOs cultured in MM (n = 3, each nine pooled hCO). iBAQ, intensity-based absolute quantification. (D) Clustered heat map of genes regulated (FDR < 0.05) between hCOs cultured in CTRL medium vs. MM. (E) Clustered heat map of proteins regulated (q value < 0.05) between hCOs cultured in CTRL vs. MM. (F) GO term analysis of RNA-seq reveals that multiple processes are decreased or increased in hCOs cultured in MM vs. CTRL medium. These are consistent with processes that occur during postnatal heart maturation (5). Numbers in the bars indicate numbers of genes identified in that particular process. (G) GO term analysis of proteomics reveals that multiple processes are decreased and increased in hCOs cultured in MM vs. CTRL medium. These are consistent with processes that occur during postnatal heart maturation (5). Numbers in the bars indicate numbers of genes identified in that particular process. (H) RNA-seq markers of maturation: genes in the cardiac development GO term, which are also up-regulated in proteomics data. (I) Proteomics markers of maturation: genes in the cardiac development GO term. Data are mean ± SEM. *P < 0.05; **P < 0.01; and ***P < 0.001, FDR (H) or q value (I) for statistical analysis (SI Methods).