Fig. 3: Metabolic maturation of cardiac organoids.

A Protocol to induce metabolic maturation in the cardiac organoids. B Left; Representative immunostaining of cTNT in the immature and mature cardiac organoids. Scale bar; 10 μm. Right; Quantification of the sarcomere length in the indicated conditions (N = 15 from 3 independent biological replicates). C Left; Representative immunostaining of Ki67 and cTNT in the immature and mature cardiac organoids. Scale bar; 20 μm. Right; Quantification of the percentage of Ki67-positive cardiomyocytes in the organoid (N = 17 from 3 independent biological replicates). D Left; Representative immunostaining of CX43 and cTNT in the immature and mature cardiac organoids. Scale bar; 20 μm. Right; Quantification of the CX43 expression in the organoid (N = 18 from 3 independent biological replicates). E Analyses of the oxygen consumption rate (OCR) in the indicated populations using the Seahorse fatty acid oxidation (FAO) assay (N = 3 independent biological replicates each). F RT-qPCR expression analyses of indicated genes in the early (2 weeks) and late (4 weeks) CD90-positive cardiac fibroblast populations isolated (FACS) from immature and mature organoids (N = 8 biologically independent samples). Adult and fetal CFs were included as a reference (N = 3 biologically independent samples each). epi: day15 epicardial cells prior to the initiation of coculture (N = 4 biologically independent samples). G Left; Representative immunostaining of fibronectin (FN) in the cardiac organoids at the indicated time points. Scale bar; 20 μm. Right; Quantification of the FN density at the indicated time points (N = 10 for 2 W, N = 30 from 3 independent biological replicates). H Left; Representative immunostaining of collagen type 1 (COL1) in the cardiac organoids at the indicated time points. Scale bar; 20 μm. Right; Quantification of the COL1 density at the indicated time points (N = 22 from 3 independent biological replicates). Statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons in (E)–(H) and by two-sided unpaired t-test in (B)–(D). All error bars represent SEM. VCM Agg ventricular cardiomyocyte aggregates. CF cardiac fibroblasts. Source data are provided as a Source Data file.