PMC

Isolation and characterization of cardiomyocytes. (A,B) FACS plots from representative experiments analysing VCAM1 and eGFP (A), and average percentages from multiple experiments of VCAM1⁺ cells (B) in the pre-isolation (grey) and post-isolation (black) fractions showing the efficiency of the isolation strategy. Experiments were performed on cells differentiated from NKX2-5^eGFP/w hESCs (upper panels, n=4) and hiPSCs (lower panels, n=6). (C) Immunofluorescence images of cardiac sarcomeric proteins TNNI (green) and α-actinin (red) in VCAM1⁺ cardiomyocytes generated from NKX2.5^eGFP/w hESCs (upper panel) and hiPSCs (lower panel). Nuclei are stained in blue with DAPI. Scale bars: 50 μm. (D,E) Representative APs at 1, 2 and 3 Hz (D), and AP parameter quantification of non-enriched (black) and VCAM1⁺ (blue) cardiomyocytes differentiated from NKX2-5^eGFP/w hESCs (upper panels, n=16 and 16) and hiPSCs (lower panels, n=15 and 18) from three independent differentiations each (E). Two-way ANOVA with Sidak's multiple comparisons test. P<0.05 versus VCAM1⁺ cardiomyocytes. Data are mean±s.e.m.

Isolation and characterization of endothelial cells. (A,B) FACS histograms from representative experiments (A) and averaged percentages from multiple experiments (B) of CD34⁺ cells in the pre-isolation (grey) and post-isolation (black) fractions showing the efficiency of the isolation strategy. Experiments were performed on cells derived from NKX2-5^eGFP/w hESCs (upper panels, n=5) and hiPSCs (lower panels, n=6). (C) Representative bright-field images of the morphological appearance of CMEC-derived CD34⁺ cells from NKX2-5^eGFP/w hESCs (upper panel) and hiPSCs (lower panel) after isolation and re-plating. Scale bars: 200 μm. (D) FACS measurement (histograms) for key endothelial cell-surface markers of CD34⁺ cells 4 days after isolation and re-plating. Specific antibody-labelled cells are shown in black (NKX2-5^eGFP/w hESC line, upper panels; hiPSC line, lower panels) (n=3). (E) qRT-PCR analysis for key endothelial genes (upper panels) and for cardiac-specific genes (lower panels) in CMEC-derived CD34⁺ cells from NKX2-5^eGFP/w hESCs (grey) and hiPSCs (black). Mann–Whitney test. *P=0.0286. n>3. Data are mean±s.e.m. Values are normalized to RPL37A. (F) qRT-PCR analysis (heatmap) and hierarchical clustering showing a panel of endothelial and cardiac genes of interest in HUAECs, HUVECs, HDBECs and HCMECs together with CMEC-derived CD34⁺ cells from NKX2.5^eGFP/w hESCs and hiPSCs (n=3). Values are normalized to RPL37A and VEC, and are relative to HUAECs.

Simultaneous induction of cardiomyocytes and endothelial cells from cardiac mesoderm. (A) The differentiation protocol towards cardiomyocyte and endothelial cell fates. Cardiac mesoderm was induced with BMP4, activin A and CHIR 99021 from day 0 to day 3, followed by treatment with XAV939 (CM), VEGF (EC) or XAV939+VEGF (CMEC). (B) Bright-field images of day 10 differentiated NKX2-5^eGFP/w hESCs under CM, EC and CMEC conditions. Scale bars: 100 μm. (C) Representative FACS plots for CD31 together with eGFP of CM, EC and CMEC populations measured in NKX2-5^eGFP/w hESCs on day 10 of differentiation. Numbers in the quadrants represent the respective percentage of cells. n=4. (D) qRT-PCR analysis at the indicated time points (d=day) for selected cardiac genes under CM (black) and CMEC (red) conditions. Values are normalized to RPL37A and relative to undifferentiated NKX2.5^eGFP/w hESCs. Two-way ANOVA with Sidak's multiple comparisons test. *P<0.05. n=3. Data are mean±s.e.m. (E) Heatmap showing qRT-PCR analysis of key genes encoding ion channels involved in AP shaping and Ca²⁺-handling proteins (linear scale). Values are normalized to RPL37A and TNNT2, and are relative to undifferentiated NKX2.5^eGFP/w hESCs. (F,G) Representative AP traces at 1, 2 and 3 Hz (F), and AP parameter quantification of day 21 NKX2-5^eGFP/w hESC cardiomyocytes differentiated under CM (black) and CMEC (red) conditions (G). Two-way ANOVA with Sidak's multiple comparisons test. Data are mean±s.e.m. n=16-24 from three independent differentiations each.

Generation and characterization of 3D cardiac microtissues. (A) The protocol to generate cardiac MTs from cardiomyocytes cultured alone (MT-CM) or in combination with enriched CD34⁺ endothelial cells (MT-CMEC). MTs from NKX2-5^eGFP/w hESCs were generated from non-enriched or enriched VCAM1⁺ cardiomyocytes, whereas MTs from hiPSCs were generated from enriched VCAM1⁺ cardiomyocytes only. MT characterization was performed between days 7 and 20 by immunofluorescence, qRT-PCR, MEAs and contraction analyses. (B) Immunofluorescence analysis of sarcomeric cardiac TNNI (green) and endothelial cell surface marker CD31 (red) of day 7 cardiac MTs from non-enriched (upper panels) and VCAM1-enriched (lower panels) cardiomyocytes from NKX2-5^eGFP/w hESCs. Percentages of CD34⁺ cells are shown at the top. Scale bars: 100 μm. (C) Immunofluorescence analyses of TNNI (green) and CD31 (red) of day 7 MTs generated from hiPSC-VCAM1⁺ cardiomyocytes. Percentages of CD34⁺ cells are shown at the top. Scale bars: 100 μm. (D) qRT-PCR analysis for key sarcomeric genes, ion channels involved in AP shaping and Ca²⁺ regulatory genes, as well as other cardiac genes of interest in day 7 hiPSC-MTs and in day 21 age-matched VCAM1⁺ cardiomyocytes from hiPSCs. All values are normalized to RPL37A and relative to undifferentiated hiPSCs. Data are mean±s.e.m., n>4. One-way ANOVA with Tukey's multiple comparisons test. *P<0.05 versus VCAM1⁺ cardiomyocytes. (E) FP representative traces measured using MEAs under baseline conditions (left panels) and upon addition of 1 μM isoprenaline (ISO) (right panels) in MT-CM (upper panels, blue) and MT-CMEC (lower panels, green) from hiPSCs. (F) QT and RR intervals measured using MEAs under baseline conditions and after increasing concentrations of ISO in MT-CM (blue) and MT-CMEC (red) from hiPSCs. One-way ANOVA. *P<0.05 versus baseline. Colour code of the asterisks indicates the experimental group. Data are mean±s.e.m., n=9. (G) qRT-PCR analysis of β-adrenoreceptors (β₁ AR, left panel; β₂ AR, right panel) in day 7 MT-CM and MT-CMEC from hiPSCs. Values are normalized to RPL37A and are relative to undifferentiated hiPSCs. Mann–Whitney test. Data are mean±s.e.m., n=3. (H,I) Representative traces of contraction (H) and contraction velocity (I) in MT-CM (blue) and MT-CMEC (green) generated from hiPSCs and paced at 0.5 (left panels) and 1 Hz (right panels). Results are shown under baseline conditions and after superfusion of 500 nM and 1 µM verapamil (VER).