For the teratoma formation assay, hESC spheres were injected directly into the testes of severe combined immunodeficiency mice (NOD.CB17-scid; CLEA Japan). After 8 weeks, the resulting teratomas were surgically dissected out of the mice and fixed with 4% PFA. The samples were embedded in paraffin, sectioned into 5 μm slices, and stained with hematoxylin and eosin. All mouse works were approved by the Institutional Animal Ethics Committee of Kyoto University.
For embryoid body formation, hPSC spheres on day 0 were cultured for 2 weeks in DMEM/F12 medium that had been supplemented with 5% KSR or 20% FBS. The medium was changed three times a week. Total RNA was purified and gene expression was analyzed by performing qRT-PCR as described above.
Induction of differentiation into cardiomyocytes or neurons during culturing was carried out as described previously (Minami et al., 2012; Sakurai et al., 2010 ). Procedures for the immunostaining analysis of the differentiated cells were also described previously (Minami et al., 2012; Sakurai et al., 2010 ). The antibodies that were used for this analysis were anti-cardiac troponin T (Santa Cruz Biotechnology), anti-αactinin (Sigma-Aldrich), and anti-NKX2.5 (Abcam) for cardiomyocytes, and anti-βIII tubulin (Sigma-Aldrich) for neurons.
For embryoid body formation, hPSC spheres on day 0 were cultured for 2 weeks in DMEM/F12 medium that had been supplemented with 5% KSR or 20% FBS. The medium was changed three times a week. Total RNA was purified and gene expression was analyzed by performing qRT-PCR as described above.
Induction of differentiation into cardiomyocytes or neurons during culturing was carried out as described previously (Minami et al., 2012; Sakurai et al., 2010 ). Procedures for the immunostaining analysis of the differentiated cells were also described previously (Minami et al., 2012; Sakurai et al., 2010 ). The antibodies that were used for this analysis were anti-cardiac troponin T (Santa Cruz Biotechnology), anti-αactinin (Sigma-Aldrich), and anti-NKX2.5 (Abcam) for cardiomyocytes, and anti-βIII tubulin (Sigma-Aldrich) for neurons.