Fig. 7: Generating mature human iPS-derived heart organoids.

a Protocol for self-assembling mature human heart organoids (hHOs) by mTOR downregulation. Created in BioRender. Lucena-Cacace, A. (2025) https://BioRender.com/9j18jf6. b MTT assay determines hHO viability and IC50 value after Torin1 treatment (96 hours, descending concentrations, 1:2 dilutions; n = 2 technical replicates). Data are presented as means ±s.d. c Human heart organoid size (mm²) before (● black, untreated) and after one-week Torin1 treatment ( purple, Torin1) (untreated at day 15, n = 5; untreated at day 15 + 7, n = 19; Torin1-treated at day 15 + 7, n = 20). Data are presented as means ± s.e.m.; two-tailed unpaired Student’s t-test. d Phase contrast microscopy of untreated and Torin1-treated hHO (scale bar: 500 µm). e Immunofluorescence images of WT1 and TNNI3 expression in hHOs treated with Torin1 (200 nM), Tacrolimus (1 µM), or Rapamycin (200 nM) (scale bar: 500 µm). f Immunofluorescence images of SNAI1 and ZO-1 expression in hHOs treated with Torin1 (200 nM), Tacrolimus (1 µM), or Rapamycin (200 nM) (scale bar: 500 µm). g schematic representation of the effects of partial (Rapamycin, Tacrolimus) and total (Torin1) mTOR inhibition in the hHO maturation h, UMAP visualization of single-cell RNA sequencing (scRNA-seq) data depicting 11 transcriptionally distinct clusters (0–10) identified based on differential gene expression profiles. Cluster 0 corresponds to epicardial cells, characterized by the expression of WT1, TCF21, and ALDH1A2. Clusters 1 and 9 represent cardiomyocytes, marked by the expression of TNNT2, TTN, MYH6, and MYH7, with cluster 1 denoted as Cardiomyocytes I and cluster 9 as Cardiomyocytes II. Clusters 2, 3, 5, 7, and 10 correspond to resident fibroblasts, expressing COL1A1, COL3A1, VIM, and THY1, with distinct subtypes labeled as Resident Fibroblasts I-IV. Cluster 6 represents activated fibroblasts/myofibroblasts, identified by high expression of ACTA2, TAGLN, MYH11, and PDGFRB. Clusters 4 and 8 correspond to endocardial cells, marked by the expression of NPR3, PROX1, NFATC1, and ESAM, with cluster 4 labeled as Endocardial Cells I and cluster 8 as Endocardial Cells II. The clustering highlights the cellular heterogeneity within the heart organoids and the presence of distinct fibroblast, cardiomyocyte, epicardial, and endocardial populations. i UMAP visualization of single-cell RNA sequencing (scRNA-seq) data displaying 13 transcriptionally distinct clusters (0–12) based on differential gene expression profiles. Clusters 0 and 12 represent cardiomyocytes, marked by the expression of MYH6, MYL7, NPPA, and NR2F2, with cluster 0 designated as Cardiomyocytes I and cluster 12 as Cardiomyocytes II. Clusters 1, 3, 5, and 8 correspond to resident fibroblasts, expressing COL1A1, COL3A1, THY1, VIM, DCN, and LUM, with subtype labels Resident Fibroblasts I-IV. Cluster 6 represents epicardial cells, characterized by the expression of WT1, TCF21, and ALDH1A2. Cluster 4 consists of endothelial cells, identified by the expression of VWF, PECAM1, CDH5, KDR, ESAM, and TIE1. Clusters 2, 7, 9, 10, and 11 represent endocardial cells, marked by NPR3, PROX1, NFATC1, ESAM, and SOX17, and are further subdivided into Endocardial Cells I-V. This clustering underscores the cellular heterogeneity within the heart organoids, revealing distinct populations of cardiomyocytes, fibroblasts, endothelial, epicardial, and endocardial cells. j Dot plot for control and k Torin1-treated hHOs illustrating the expression patterns of key marker genes across identified clusters, enabling cell type annotation based on characteristic gene signatures. The size of each dot represents the proportion of cells within a cluster expressing the given gene, while the color intensity reflects the average expression level. l Heatmap displaying the log fold-change (logFC) of differentially expressed genes (DEGs) related to cardiomyocyte maturation across identified clusters. The color scale represents the relative expression levels m Heatmap displaying the log fold-change (logFC) of differentially expressed genes (DEGs) related to epicardial maturation across identified clusters. The color scale represents the relative expression levels n Bar graph illustrating the expression levels of key endothelial marker genes in Cluster 4 following Torin1 treatment in human heart organoids (hHOs). Expression values are normalized and presented as mean expression per cell, highlighting the relative abundance of endothelial-specific genes. The analysis reveals differential regulation of endothelial markers in response to Torin1 treatment. Source data is provided as a Source Data file.