Gene Expression Analysis of hESC Differentiation Figure 1. Characterization of undifferentiated and differentiated hESCs toward neural phenotype. Schematic representation of neural differentiation procedure and stages NI, NE, NT and DN in the upper panel. (A) Phase contrast photomicrographs of a colony of hESCs grown under feeder-free conditions. The cells show a high ratio of nucleus to cytoplasm and a compact morphology with clear borders. Immunofluorescence analysis of key hESC markers, including OCT4 (B) and NANOG (C) expression, in hESCs. The hESCs were induced to differentiate into neural ectoderm by RA, SHH, Noggin and LIF, and the differentiating cells, after 9 days of treatment with extrinsic factors, changed their morphology and formed rosette type structures (D). Immunostaining for the neural markers Nestin (E), SOX1 (F) and Pax6 (G) and flowcytometry results for neural progenitor’s marker genes (H) revealed the differentiation of hESCs toward neural cells. Further differentiation of rosette cells in the absence of Noggin, Shh, and RA for an additional week resulted in NT formation. NTs were then dissected manually (I) and, after dissociation, were replated onto laminin/poly-L- ornithine-coated dishes (J) for an additional two weeks in neurobasal medium supplemented with N2 and BDNF, GDNF, AA and cAMP to form a pool of matured neural cells (K). Immunostaining was performed and indicated the presence of differentiated neurons (L), astrocytes (M), serotonergic neurons (N) and dopaminergic neurons (O). Nucleuses are stained with DAPI and PI (propidium iodide). RA: Retinoic acid, Shh: Sonic hedgehog, LIF: Leukemia inhibitory factor, BDNF: brain derived neurotrophic factor, GDNF: Glial derived neurotrophic factor, AA: Ascorbic acid, NI: Neural induction, NE: Neural ectoderm, NT: Neural-like tube, DN: Differentiated neural cells. doi:10.1371/journal.pone.0022856.g001 PLoS ONE | www.plosone.org 4 July 2011 | Volume 6 | Issue 7 | e22856