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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.
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