Fig. 1: Three-step WNT signaling modulation triggers heart organoid formation in 3D culture. | Nature Communications

Fig. 1: Three-step WNT signaling modulation triggers heart organoid formation in 3D culture.

From: Self-assembling human heart organoids for the modeling of cardiac development and congenital heart disease

Fig. 1

a A schematic diagram depicting the protocol used to differentiate TNNT2 + cardiomyocytes in embryoid bodies. CHIR99021 concentration is variable at day 0 and day 7. (Blue cells represent PSCs, red cells represent cardiomyocytes, all other colors represent non-myocyte cells). b Brightfield images of developing organoid over 15 days of differentiation (top), confocal immunofluorescence images for DAPI (blue), and TNNT2 (red), of representative organoids by day from day 0 to 15; scale bar: 500 µm, inset: 50 µm (n = 20). c Confocal immunofluorescence images for DAPI (blue) and TNNT2 (red), in day 15 organoids differentiated using 4 µM CHIR99021 showing sarcomere bands; Scale bar: 25 µm. d Cell quantification of cardiomyocytes within organoids taken at multiple z-planes as a percentage of TNNT2 + cells to total cells of each organoid for the five CHIR99021 concentrations (n = 5 organoids). Value = mean ± s.d., 1 way ANOVA multiple comparison test; *p = 0.02, ****p < 0.0001, ns: no significance p = 0.08. e Percentage of TNNT2 + cells normalized to total cells in confocal images of hHOs (n = 20 organoids), and f beating frequency (n = 7 organoids), in 3 iPSC lines and 1 ESC line. Value = mean ± s.d. g Confocal immunofluorescence images of hHOs at differentiation day 15 for DAPI (blue), WT1 (green), TNNT2 (red), and TJP1 (white) after day 7 epicardial induction with 2 µM CHIR99021 (n = 12 organoids). h Cell quantification of cardiomyocyte (TNNT2 +) and epicardial cells (WT1 + and TJP1 +) within organoids taken at multiple z-planes as a percentage of total cells of each day 15 organoid (n = 5 organoids). Value = mean ± s.d., 1 way ANOVA multiple comparison test; *p = 0.04, **p = 0.0023, ****p < 0.0001, otherwise ns: no significance p = 0.9. E8: Essential 8 media, RPMI + B27 – ins: RPMI with B27 supplements without insulin. i Sketch of hHOs showing surface (top) and cross-section (bottom) features of the hHOs. Source data are provided as a Source Data file.

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