FIG. 1.

Time-course analysis of the transcriptional profiles of genes associated with both pluripotency and early differentiation in GSKi-treated hESCs. (A) H1-hESCs were treated with 5 μM CHIR99021 using STEMdiff APEL as the basal differentiation media without additional growth factors/cytokines under feeder- and serum-free conditions. Subsequently, RNA was extracted from GSKi-treated hESCs at 24 h intervals from day 0 to 5, and quantitative real-time PCR (qRT-PCR) analysis was performed using primers specific to PS (T, MIXL1, GSC, and FOXA2), pluripotency (OCT4, NANOG, and SOX2), mesoderm (KDR and PDGFRα), ectoderm (SOX1 and PAX6), and endoderm (CXCR4 and SOX17) genes. The transcript levels of PS-associated genes were up-regulated in just 24 h of GSKi treatment and were subsequently down-regulated thereafter. All qRT-PCR data were normalized to β-actin as an endogenous control and were presented as expression levels (fold) relative to day 0, with undifferentiated hESCs as controls. (B) T (brachyury) and OCT4 expression was analyzed by immunofluorescence microscopy. T was visibly detected in hESCs after 24 h of GSKi treatment and began to down-regulate by day 2. (C) β-catenin expression in hESCs after GSKi treatment. Top row: β-catenin expression in undifferentiated hESCs, middle row: after 24 h of GSKi treatment, bottom row: no-GSKi control. All scale bars represent 200 μm. GSK, glycogen synthase kinase; GSKi, GSK inhibitor; hESCs, human embryonic stem cells; PS, primitive streak; GSC, Goosecoid. Color images available online at www.liebertpub.com/scd