Differentiated and WY-14643 treated cells form organoids. (A) CDCs, expanded on ColIV were plated on FN and differentiated by treatment with 5-Azacytidine (5-Aza) for 3 days followed by addition of ascorbic acid (AA) or a combination of AA/TGFβ/WY-14643 as indicated for 30 days. (B) Representative micrographs of WY-14643-treated differentiating CDCs which started to aggregate on day 10 and formed organoid/tubular structures on day 20. Scale bar 100 µm (C) Z-stack confocal images of 3D organoids showing expression of MHC, Titin, cTnnT2, MLC, CX43 as indicated, scale bar = 300 µm, unmerged images are shown in Figure S11. (D) The relative mRNA expression of cardiac genes was assessed using qPCR. The mRNA expression was normalized to the geometric mean of Actb and β2M as reference genes and baseline as calibrator; Green stars indicate comparison between baseline and differentiated cells and black stars indicate comparison between differentiated cell groups; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)