Fig. 1. (A) Visualization of visceral endoderm differentiation in ES cell aggregates by using GFP expression under the control of the promoter of the Afp gene (which encodes -fetoprotein). GFP expression was not detected when Afp-GFP ES cells were cultured as monolayer in the presence of LIF (ES, left). GFP-positive cells appeared on the outer layer of aggregates within 4 days regardless of the presence of LIF in the medium (LIF– or LIF+, middle and right). (top) Phase-contrast image under a light microscope; (bottom) pictures under a fluorescence microscope. Bars, 200 µm. (B) Expression of ES-cell-specific genes and differentiation markers upon ES-cell aggregation. Total RNA was isolated from undifferentiated ES cells (ES) and ES-cell aggregates (Day2, Day4). RNA was subjected to RT-PCR analysis and the PCR products were separated on a 2% agarose gel and visualized by ethidium-bromide staining. TTR, transthyretin. (C) The LIF/STAT3 pathway and BMP4 stimulation did not inhibit visceral endoderm differentiation upon ES-cell aggregation. Total RNA was isolated from STAT3ER ES cells, which were maintained as monolayer cells in the medium containing either LIF or 4HT (ES) and the cells, which were cultured as aggregates for 2 days in the same medium with or without BMP4 as indicated.