Fig. 1.
a ES cell in vitro neuralization. DIV days of in vitro differentiation. 0DIV corresponds to the time of leukemia inhibitory factor (LIF) withdrawal. N2 and B27 are the supplements used in the minimal medium of differentiation. Example of bright-field microphotographs of cells at different DIV are shown on the bottom. ELA epiblast-like aggregates, NPC neural progenitor cells, NPC/Neu neural precursors, Neu differentiated neurons. b RT-PCR gene expression analysis. Values are relative to β-actin mRNA expression. Highest and lowest expression levels were normalized to 1 in the left/middle histograms and in the right histogram, respectively. c, d Oct4 and Nanog immunodetection in ES cells (c) or ELA cells (d). e Violin plot shows the distribution of green fluorescent protein (GFP) intensity in a TNG-A Nanog::GFP line [16] in LIF/serum (ES cells, red) and 24 h (green) or 48 h (blue) after LIF/serum withdrawal (ELA) or Activin/fibroblast growth factor (FGF)2 induction (EpiSC), respectively. f, g Derivation of epiblast stem cells (EpiSC) and ELA-EpiSC from ES and ELA cells, respectively. h, i EpiSC and ELA-EpiSC bright-field images. j Expression correlation of markers of pluripotency and priming between EpiSC (y-axis) and ELA-EpiSC (x-axis). Values are expressed as log2ΔCt of RT-PCR assay; R 2 coefficient of determination. k Hierarchical clustering analysis on Spearman correlation between different microarray samples. l Flow cytofluorimetric analysis of Sox1::GFP cells (46C line), indicating the ratio of GFP-positive cells (y-axis) in different cell types or times of differentiation (x-axis). m, n Immunodetection of neural markers at 7 days of ELA-EpiSC neuralization. o RT-PCR gene expression analysis as in b in ELA-EpiSC after 4 (+4DIV) or 8 (+8DIV) days from FGF2/Activin A withdrawal. Error bars in b, l, and o show standard error. In b and o *p = 0.05, **p = 0.01 (REST randomization test). Scale bars are 30 microns in a, c, and d, 40 microns in h, i, m, and n