Characterization of 253G1 cells subcultured on laminin-521 in Essential 8 medium.

(A) Expression levels of undifferentiated cell markers (OCT3/4, NANOG, SOX2 and LIN28) in 253G1 cells subcultured on laminin-521 in Essential 8 were determined using qRT-PCR. Relative mRNA expression levels are presented as ratios to the level of that in control cells on Matrigel. Results are the mean ± SD (n = 3). (B) In vitro differentiation analysis of 253G1 cells subcultured on laminin-521 in Essential 8 medium. Immunostaining of the markers for three germ layers are shown: endoderm (alpha-fetoprotein (AFP)), mesoderm (α-smooth muscle actin (SMA)) and ectoderm (βIII tubulin). Scale bars, 200 µm. (C) Expression levels of differentiated cell markers in embryoid bodies (EBs) derived from 253G1 cells: endoderm (GATA6, SOX17), mesoderm (CDH5, FOXF1), ectoderm (SOX1, PAX6). Relative mRNA expression levels are presented as ratios to the level of that in control cells (EBs at Day 10). Results are the mean ± SD (n = 3). (D-E) Teratomas derived from 253G1 cells cultured on laminin-521 in Essential 8 medium are shown. Hematoxylin and eosin staining showed the features of three germ layers: Ep, epithelium-like tissue (endoderm); Ca, cartilage (mesoderm); Ne, neural rosette-like tissue (ectoderm); P, pigmented neuroectodermal resembling meranocyte (ectoderm); Br, brain-like tissue (ectoderm). Scale bars, 200 µm.