Fig. 1. Fabrication of human cardiac tissues with a heart extracellular microenvironment.

a Schematic illustration of human cardiac tissue fabrication using three cell types, heart extracellular matrix (HEM) hydrogel, and a microfluidic chip. b Hematoxylin and eosin (H&E) staining and DNA content, c Alcian blue staining and glycosaminoglycan (GAG) content, and d Masson’s trichrome (MT) staining and collagen content of native and decellularized porcine heart tissues (scale bars = 50 μm, N = 3, biological replicates, ***p < 0.001). e–g Proteomic analysis of decellularized porcine tissue-derived extracellular matrix (ECM) samples. e ECM composition and the 10 most abundant matrisome proteins in porcine HEM hydrogel samples derived from different batches (A#1, A#2, and A#3) and different donors (A, B, and C) (N = 3, biological replicates). Red letters represent proteins that overlapped between different batches or donors. f Principal component analysis and Pearson’s correlation analysis of total proteins contained in different HEM samples (A#1, A#2, A#3, B, and C). The mean values of three biological replicates were used for analysis. g Percentage of heart tissue-enriched proteins (4-fold higher than in other tissues) among total proteins contained in the porcine HEM A#1 sample (left) and Gene Ontology Biological Processes (GOBP) analysis of the enriched proteins (right). Fisher’s exact test with false discovery rate correction was used for data analysis. h Elastic moduli of HEM hydrogels produced at concentrations of 2, 4, and 6 mg/ml (N = 4, biological replicates, **p < 0.01 and ***p < 0.001). i The projected areas of cardiac tissues fabricated using 2, 4, and 6 mg/ml HEM hydrogel (N = 8, biological replicates). j Immunofluorescent images of cardiomyocyte (CM; cTnT), endothelial cell (EC; CD31), and cardiac fibroblast (CF; VIM) markers in each group of cardiac tissues (scale bars = 200 μm). k Relative mRNA expression levels of CM (MYH7, TNNT2, and NPPA), EC (PECAM1, vWF, and CDH5), and CF (COL1A1 and PDGFRA) markers in each group of cardiac tissues (N = 4, biological replicates, *p < 0.05, **p < 0.01, ***p < 0.001, data are representative of two independent experiments). Cardiac tissues prepared using human induced pluripotent stem cell (hiPSC)-derived CMs, CFs, and human umbilical vein endothelial cells (HUVECs) in (i–k) were cultured for 7 days and used for analysis. Data are presented as means ± S.D. Statistical significance was determined using unpaired two-sided Student’s t-tests in (b–d) and one-way ANOVA with Tukey’s multiple comparisons tests in (h and k). Non-significant statistical differences are indicated as n.s. (p > 0.05).