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Fig. 2. Bmp4 induces mesodermal specification. (A) GFP-Bry ES cells
were differentiated in serum-free media either with serum or with various
concentrations of Bmp4 added at the start of the culture (day 0). At the
indicated time point in days, the percentage of live cells positive for GFP
was determined by flow cytometry. (B) Gene expression analysis
performed on ES cells and day 1.5, 2.5 or 3.5 serum-free plus Bmp4 EB-derived
cells by RT-PCR. β-actin expression levels were used for cDNA quantity
control. (C) Cells derived from day-3.5 EBs were assessed for their
relative percentage of GFP-Bry and Flk1 expression. (D) Cells derived
from day-3.5 EBs were assessed for their ability to form blast and VSM
(vascular smooth muscle) colonies upon secondary replating. Colonies were
scored 4 days after replating. *A statistically significant
difference (P<0.05) compared with the serum control. Data are
presented as the mean number of colonies from three dishes. Error bars
represent s.e.m. (E) Tie2 and CD45 expression levels were analyzed on
pools of replated colonies derived from day-3.5 EBs stimulated with Bmp4.
(F) Morphology of VSM colonies in secondary replating at day 4 of
culture (20x magnification). Individual colonies were expanded on glass
coverslips for 8 days and stained for the expression of CD31 (red) and smooth
muscle actin (green); nuclei are stained in blue with DAPI (20x
magnification). (G) Individual VSM colonies were cultured for 4 days in
Matrigel plugs (10x magnification). All data shown are representative of
at least three experiments.
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