FIG. 2.

A PS/mesendoderm-like cell population exists in the initial stages of GSKi treatment as analyzed by FACS. (A) The representative morphology of the hESC colonies under GSKi treatment at the indicated time points was shown. Scale bars: 500 μm for 10× magnification (top row), 1 mm for 4×(bottom row). (B) Time-course FACS analysis in GSKi-treated hESCs. Cells were harvested at different time points (days 0, 1, 2, 4, and 7) and stained with KDR-APC, PDGFRα-FITC, and CXCR4-PE for FACS analysis. Cells expressing CXCR4 were detected starting at day 2 of GSKi treatment, which is also concurrent with the morphological spreading of cells from the edges of the hESC colony. Prolonged inhibition of GSK-3 eventually resulted in a pure CXCR4⁺ cell population. (C) Analysis on the mesoderm potential of hESCs after GSKi treatment. Cells were treated with BMP4 for 24 h after different durations of GSKi treatment as indicated: G1B, G2B, G4B, and G7B, the number representing the days of GSKi treatment. For the G0B group, hESCs were differentiated directly in BMP4 for 48 h without prior GSKi treatment. FACS, fluorescence-activated cell sorting; APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin; BMP, bone morphogenetic protein.