Fig 2: Characterization of cardiomyocytes derived from WTSIi020-A and UKKi025-A. (A,B) Flow cytometry analysis of cells positive for TNNT2 and OCT4 at the beginning (day 0) and the end (day 15) of differentiation in CDM3. (C,D) Median fluorescence intensities of TNNT2+ and OCT4+ populations at day 0 and 15 of the CDM3 protocol. n = 3 biological replicates. (E,F) Heatmap plots representing RT-qPCR for markers of pluripotency (NANOG, OCT4, SOX2, KLF4, MYC), cardiac progenitors (NKX2.5) and cardiomyocytes (TNNT2, MHY6, MHY7) on day 0 and day 15. n = 3 independent biological replicates performed in technical triplicates. (G) Immunofluorescent staining images of cardiomyocytes derived from WTSIi020-A and UKKi025-A. WTSIi020-A CMs show clear formation of immature sarcomeric-like structure while UKKi025-A CMs display a disrupted structure and rather enlarged cell morphology (marked with *). Significance level A: **** poct4 < 0.001; * ptnnt2 < 0.023; B: *** poct4 = 0.0002; **** ptnnt2 < 0.0001; C: ** poct4 = 0.0015; **** ptnnt2 < 0.0001; D: * poct4 = 0.0142; **** ptnnt2 < 0.0001; E: p = 0.0001; F: p = 0.0002.

Fig 3: Determining the structural development of cardiac organoids. (A–C) Immunofluorescence images of Cor-Oids, WTS-Oids and UKK-Oids after 21 days of culture, labelled with cardiac-specific troponin (TNNT2), vascular cadherin (VeCADH) and vimentin (VIM). We identified similar structural patterns (A–C, iii–iv) in all organoid types, in terms of HCMEC (magenta) and HCF (green) distribution. Different structural development patterns were observed regarding the migration of hiPSC-CMs and the formation of cardiac filament-like structures (red), which were well-defined in Cor-Oids (A, i–ii), mostly perimetric in WTS-Oids (B, i–ii), and still in formation in UKK-Oids (C, i–ii). Scale bars = 200 µm.

Fig 4: Transcriptomic changes across in vitro cardiac differentiation. A. Significant batch and line variations in cTnT+ cells at day 15 of in vitro cardiac differentiation. B. Experimental workflow to perform scRNA-seq sequentially on hiPSCs during different stages of in vitro cardiac differentiation from one differentiation each for hiPSC 1 and 2. C. UMAP representation of the scRNA-seq dataset with superimposed colours representing different cell clusters and a black arrow indicating the single cell trajectory of transcriptomic changes in in vitro cardiac differentiation. D. Single cell heatmap on the representative DEGs expressed in each cluster. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig 5: In vitro APLNR+ cardiac progenitors to differentiates into cardiomyocytes and cardiac mesenchyme. A. Experimental workflow to verify in vitro APLNR+ cardiac progenitors and its lineage descendants. B. Increased and consistent 75–80% cTnT+ in day 10 APLNR+ cell fraction. Data represented as mean ± SD from three independent differentiations for each hiPSC line. *P < 0.05, **P < 0.01, ***P < 0.001 C. Representative cTnT flow cytometry scatter dot-plots to illustrate Fig. 4B. D. Increased expression of cardiac progenitor markers in day 5 APLNR+ cell fraction. Data represented as mean ± SD from three independent differentiations for each hiPSC line. *P < 0.05, **P < 0.01, ***P < 0.001 E. Increased expression of cardiomyocyte and cardiac mesenchyme markers in day 10 APLNR+ cell fraction. Data represented as mean ± SD from three independent differentiations for each hiPSC line. *P < 0.05, **P < 0.01, ***P < 0.001 F. Immunocytochemistry showing increased expression of TNNT2 in day 10 APLNR+ cell fraction.