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. 2012 Aug 15;139(16):2866–2877. doi: 10.1242/dev.078519

Fig. 5.

Fig. 5.

mESCs from 2i and LIF conditions can give rise to cXEN cells, unlike EpiSCs. (A) Representative phase-contrast images of mESCs from 2i and LIF conditions or EpiSCs at defined time-points during the cXEN protocol (day 0 in pluripotency conditions, day 3 prior to the first passage, day 9 after the second passage and day 35) (representative image of three biological replicates of stem cell lines from 2i and LIF conditions or two biological and six technical replicates of EpiSCs). Scale bars: 50 μm. (B) Immunofluorescence analysis of cXEN cells from 2i and LIF or differentiated cells from EpiSCs at day 35 of the protocol. Cells were immunostained and analyzed for the expression of Oct4, laminin, Dab2, Sparc, Gata4, Gata6, Sox17 and Sox7 expression (red), and DAPI (blue) merge. Scale bars: 100 μm. (C,D) qRT-PCR analysis of cXEN cells from 2i and LIF conditions or differentiated cells from EpiSCs for the expression of XEN-associated genes (C) or pluripotency-associated genes (D). Relative expression reflected as a fold difference compared with embryo-derived XEN or mESCs (=1), respectively. Data are the mean±s.e.m. Three biological replicates (from 2i and LIF) or two biological and three technical replicates (from EpiSCs). (E) qRT-PCR analysis of differentiated cells from EpiSCs. Relative expression reflected as a fold difference (log scale) compared with mESCs (=1) for germ cell lineage-associated genes: Sox1 and Olig3 (ectoderm); Pdx1 and Afp (endoderm); Sma, Sm22a, Smmhc and Nkx2.5 (mesoderm). Data are the mean±s.e.m. Two biological and three technical replicates. *P<0.05; **P<0.01; ***P<0.001.