FIGURE 3

Transgene expression throughout differentiation of transduced hES cells in vitro. (A) FACS analyses of transduced hES cells that were cultured for 1 and 4 weeks without passage. The percentages of EGFP-expressing cells are indicated together with the mean fluorescence intensity (in parentheses). (B, C) Phase-contrast (B) and fluorescence microscopy (C) images of cystic EBs, 20 days after generation from transduced hES cells. (D-H) Immunophenotyping of differentiated cells from disaggregated cystic EBs demonstrating co-expression of EGFP (green fluorescence) and indirect immunofluorescence staining (red) for the mesodermal markers muscle actin (D) and desmin (E), and the endodermal markers -fetoprotein (F), laminin (G), and LMW cytokeratin (H). (I-J) Phase-contrast (I) and fluorescence microscopy (J) images of neural progenitor spheres generated from transduced hES cells. (K-R) Indirect immunofluorescence analyses of neural progenitors and their differentiated progeny demonstrating co-expression of EGFP (green) and markers (red) of the neural progenitors nestin (K), PSA-NCAM (L), and A2B5 (M); early neurons (-tubulin III (N)); mature neurons (MAP 2a,b, (O) glutamate (P), NF-160 (Q)); and astrocytes (GFAP (R)). Arrows indicate cells co-expressing EGFP and markers. Scale bars represent 100 m (B, C, I, J), 20 m (D-H), and 10 m (K-R).