Figure 2

Developmental properties of CPVT-iPSC confirm their pluripotency. (A) Phase-contrast (Phc) image of EBs from CPVT-iPSC at day 6 after formation. Immunostaining of differentiated CPVT-iPSC showing EBs containing cells representative of each of the three embryonic germ layers: endoderm (α-fetoprotein for intestinal cells), ectoderm (βIII tubulin for neuronal cells) and mesoderm (α-smooth muscle actin for skeletal muscle, α SMA); nuclei were stained with DAPI. Scale bars=100 μm; (B) semiquantitative real-time PCR of differentiated control- (WT) and CPVT-iPSC at days 30 and 50 of differentiation, showing upregulation of expression of markers of the three germ layers: positivity for NCAM1, βIII-tubulin and KRT14 was indicative of ectodermal cells (neurons or epidermis); the presence of DESMIN and PECAM1 indicated the presence of mesodermal cells; and the transcription factors GATA6 and SOX17 were indicative of endodermal differentiation. Data are presented relative to undifferentiated iPSC and were normalized to HGPRT (hypoxanthine–guanine phosphoribosyltransferase) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase). Values are mean±S.D. *P<0.05; (C) teratoma formation assay: hematoxylin–eosin staining (a–c) and immunohistochemistry (d–f) of teratomas formed from CPVT-iPSC (representative images from one cell line), showing differentiation of cells injected in vivo into various tissues derived from all the three germ layers: retinal epithelium and neural rosettes derive from ectoderm (d); cartilage and muscle (positivity for α-actinin) are mesodermal tissues (e); whereas the presence of respiratory and intestinal (cytokeratin-20 (CK-20) positive) epithelium is indicative of endodermal differentiation (f)