Fig. 4. Induction of mesoderm and endoderm derivatives in EBs differentiated in the presence of activin A. (A) Left panel: comparison of the hematopoietic progenitor potential of EBs differentiated in the presence of activin and serum. D5100, day five EBs differentiated in SF cultures in the presence of 100 ng/ml activin; D6 serum, day 6 EBs differentiated in the presence of serum. Right panel: hematopoietic progenitor potential of activin-stimulated serum-free EBs (day 5 or 6) following an additional 3 days of culture in serum containing medium (+transfer). EBs were generated in SF cultures in either 0 ng/ml, 3 ng/ml or 100 ng/ml of activin. Numbers represent colonies per 5x10⁴ cells plated. Data represents mean±s.e.m. (n=3). (B) Expression analysis of cultures from EBs differentiated in the presence of different activin concentrations. Day 6 EBs differentiated in variable concentrations of activin were transferred into SF media without activin for 4 days and then replated in serum hepatocyte conditions for an additional 4 days. At day 14, replated EBs were harvested and analyzed by RT-PCR. Prior to harvesting, the proportion of EBs with visible skeletal muscle outgrowth was evaluated (indicated below panel). Numbers on top of panel indicate the activin concentration used (ng/ml). (C) Immunostaining demonstrating expression of skeletal myosin and -actinin in skeletal muscle outgrowths generated from EBs differentiated in the presence of 3 ng/ml activin. EBs were generated as in section (B) above. At day 10, EBs were plated on gelatin-coated coverslips, cultured for 4 days and then stained with antibodies to skeletal myosin and -actinin. (D) Expression analysis of GFP-Bry⁺ and GFP-Bry^– populations isolated from EBs differentiated for 5 days in the presence of 3 ng/ml or 100 ng/ml of activin. Cells from the pre-sorted, the GFP-Bry⁺ and GFP-Bry^– populations were reaggregated in SF cultures in the absence of activin for 8 days. At day 13, the reaggregated EBs were replated in hepatocyte conditions for 4 days and then harvested for RT-PCR analysis.