Fig. 5: BMP4 and Activin A improve heart organoid differentiation and development.

a–g All panels compare hHOs differentiated with CHIR99021 alone (control) and with CHIR + BMP4 + Activin A (treated). a Percent of cardiomyocyte and epicardial positive cells as a percentage of total DAPI + nuclei (n = 7 organoids per condition, value = mean ± s.d., 2-way ANOVA Sidak’s multiple comparisons test, ns: p = 0.76 for TNNT2 and p = 0.97 for WT1 + TJP1) and b organoid diameter, (n = 13 organoids per condition, value = mean ± s.d., two-tailed, unpaired t-test, *p = 0.012). c Dashed lines showing the diameter of a control (left) and treated (right) organoid averaged to determine the diameter. d Interconnectivity of chambers measured by their separation by thin TNNT2 ⁺ filaments or by thin channels showing clear connection (n = 10 organoids per condition, value = mean ± s.d., two-tailed, unpaired t-test, *p = 0.041). e immunofluorescence images of hHOs showing interconnected chambers (yellow arrows), TNNT2 ⁺ filaments (white arrows), and channels connecting chambers (green arrows), DAPI (blue), TNNT2 (red), scale bar: 500 µm, inset: 100 µm. f PECAM1 ⁺ cells as a percentage of total DAPI + nuclei, (n = 7 organoids per condition, value = mean ± s.d., two-tailed, unpaired t-test, **p = 0.0023). g Immunofluorescence images of hHOs showing DAPI (blue), PECAM1 tissue (green), and TNNT2 tissue (red), scale bar: 500 µm, inset: 50 µm (n = 12).