Figure 2

Sequential induction of hCBiPSCs towards dopaminergic neurons. (A) Schematic summary of the differentiation procedure. Phase-contrast images (B-F) and immunocytochemical stainings (G-K) during in vitro differentiation. Oct4-positive hCBiPSC colonies (B,G) were detached from the feeder layer and cultured in suspension as embryoid bodies (EBs) in the presence of dorsomorphin (DM) and SB 431542 (SB) for six days. During this time Oct4 expression was completely lost, whereas cells started to express the neural stem cell marker Pax6 (H). On day 4, purmorphamine (PMA) and FGF8 were added to initiate regionalization. After 12 days the vast majority of EBs coexpressed the midbrain marker FoxA2 (I). EBs were plated on PLO/laminin-coated cell culture dishes on day 12. Tuj1-positive neuronal cells spread out (J) and maturated into numerous dopaminergic (DA) neurons in the presence of BDNF, GDNF, TGFβ3, cAMP and ascorbic acid as indicated by tyrosine hydroxylase (TH)-positive cells (K). Scale bars represent 100 μm. FGF8, fibroblast growth factor 8; hCBiPSC, human cord blood induced pluripotent stem cells.