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Fig. 3. Activin A and bFGF induce hemangioblast specification. (A)
VEGF, activin A or bFGF were added at day 2.5 of serum-free culture
supplemented with Bmp4 at 4 ng/ml from day 0. EBs were harvested at day 3 and
assessed for their ability to form blast colonies by secondary replating.
(B) activin A and bFGF were added at day 2.5 of serum-free culture
supplemented with Bmp4 at 4 ng/ml from day 0. EBs were harvested 3, 6 or 12
hours after the induction with activin A and bFGF and assessed for their
ability to form blast colonies by secondary replating. *A
statistically significant difference (P<0.05) compared with
Bmp4-induced cultures. (C) Tie2, CD45, CD31 and VE-cadherin expression
levels were analyzed on pools of blast colonies. Top panel presents CD45 and
Tie2 staining; the level of CD31 and VE-cadherin expression was then assessed
on CD45+Tie2- (middle) and
CD45-Tie2+ (bottom) cells. (Da-c) Individual
blast colonies (examples a, b and c) were tested for their potential to
generate endothelium tubule-like structures in Matrigel plugs. Colony in c is
stained with CD31 antibody. 10x magnification, bright field. (E)
At 3, 6 and 12 hours after activin A and bFGF stimulation, cells were tested
for their relative expression of GFP-Bry and Flk1 by flow cytometry.
(F) EBs were grown for 2.5 days in serum-free culture with 4 ng/ml Bmp4
and then activin A and bFGF were added. At days 4, 5 and 6 of differentiation,
EB-derived cells were tested for the presence of hematopoietic progenitors by
colony-forming ability in secondary replating assay. Primitive, primitive
erythrocytes; definitive, all definitive colonies (see
Fig. 1). Primitive colonies
were scored 5 days after replating, definitive colonies after 8 days.
**A statistically significant difference (P<0.05)
compared with the serum control. All data shown are representative of at least
three experiments. For A, B and D, data are presented as the mean number of
colonies from three dishes. Error bars represent s.e.m.
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