Fig. 2. Endoderm potential of GFP-Bry⁺ cells. (A) FACS profile of day 2.5 EBs and reaggregation and culture protocol for differentiation of the sorted cells. (B) RT-PCR expression analysis of pre-sorted (p), GFP-Bry^– and GFP-Bry⁺ cells. d2.5, cells analyzed immediately after isolation by sorting; d6.0, reaggregated EBs; d10, cells from replated EBs. (C) Immunostaining of GFP-Bry⁺ and GFP-Bry^– cells. Upper panels shows day 2.5 EB sorted cells stained with an antibody to Foxa2. Positive cells are red/pink in color. Nuclei of all cells are stained with DAPI (blue). Bottom panels show cells from 10-day old cultures stained with an antibody to albumin. Positive cells are indicated by red color. (D) RT-PCR expression analysis of genes associated with liver maturation in populations derived from GFP-Bry⁺ and GFP-Bry^– day 2.5 EB cells. Cells from the pre-sorted population as well as those from the GFP-Bry⁺ and GFP-Bry^– fractions were reaggregated and cultured in SF conditions for 8 days. At this stage, the reaggregated EBs were replated into serum hepatocyte conditions for 4 days, harvested and analyzed.