a Immunofluorescent staining to detect cTnT (a CM marker, green)/αSMA (an SM marker, red) and b PECAM (an EC marker, red) in the HO and E10.5 heart cryosections. Expression of cTnT and αSMA was observed in the HOs (n = 7 out of n = 9, two experiments). Also, segregated lining expression of PECAM (red) was detected in the HOs (over n = 9, two experiments). Scale bar: 100 μm. c–f Immunostaining to detect c GATA4 (red)/cTnT (green), d Cx43 (red)/Cx40 (green), e Cx43 (red)/Cx45 (green), and f AFP (red)/cTnT (green). Scale bar: 50 μm. c Expression of GATA4, a cardiac TF, was observed in both HOs (n = 8 out of n = 9, two experiments) and E12.5 heart. d Expression of Cx40 in atrium-like region detected in the HOs (n = 7 out of n = 13, two experiments) was similar to that of atrium in E13.5 heart. e The organized distribution of two types of Connexin (Cx43 and Cx45) in the HOs (over n = 8, two experiments) was similar to that of E12.5 heart (the ventricle part), although a small number of Cx45 (green)-positive cells was observed. f No expression of AFP (a liver marker) was detected in both HOs (n = 8, over two biologically independent experiments) and E12.5 heart.