First published online May 17, 2004
doi: 10.1242/10.1242/dev.01130
Development 131, 2749-2762 (2004)
Published by The Company of Biologists 2004
A hierarchical order of factors in the generation of FLK1- and SCL-expressing hematopoietic and endothelial progenitors from embryonic stem cells
Changwon Park1,2,*,
Iva Afrikanova1,*,
Yun Shin Chung1,
Wen Jie Zhang1,
Elizabeth Arentson1,
Guo hua Fong3,
Alexander Rosendahl4 and
Kyunghee Choi1,2,
1 Department of Pathology and Immunology, 660 South Euclid Avenue, Campus Box
8118, St Louis, MO 63110, USA
2 Developmental Biology Program, Washington University School of Medicine, 660
South Euclid Avenue, Campus Box 8118, St Louis, MO 63110, USA
3 Center for Vascular Biology, University of Connecticut Health Center, CT
06030, USA
4 Department of Biosciences, AstraZeneca R&D Lund, Sweden
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Fig. 1. BMP4 induces FLK1+ cells. (A) ES cells were differentiated in
serum (FCS) (upper panel) or in serum-free conditions (SR) with BMP4 (5
ng/ml), BMP2 (5 ng/ml), bFGF (10 ng/ml), activin A (2 ng/ml) or TGFß1 (1
ng/ml). At day 2.75-3 of differentiation, EB cells were FACS analyzed for FLK1
expression. Numbers in insets indicate the percentage of FLK1+
cells. 2AB indicates cells stained with secondary antibody alone. (A')
Results from four independent experiments are shown as a percentage of BMP4
control. Error bars indicate s.e.m. (B) ES cells were differentiated in serum
(FCS) alone or with Noggin (upper panel). Alternatively, ES cells were
differentiated in serum-free conditions (SR) with BMP4 alone or with BMP4 and
Noggin (lower panel). At day 2.75-3 of differentiation, EB cells were FACS
analyzed for FLK1 expression. Numbers in parenthesis indicate Noggin
concentration (µg/ml). IgG1 isotype antibodies were used as control.
Numbers in insets indicate the percentage of FLK1+ cells.
(B') Results from three (FCS) or five (serum-free conditions)
independent experiments are shown as a percentage of serum alone (FCS) or BMP4
(SR) control. Error bars indicate s.e.m. (C) ES cells were differentiated in
serum-free conditions, and then BMP4 was added at different days (D0, D1, D2,
D3 or D4). At day 6 of differentiation, EB cells were FACS analyzed for FLK1
expression. Numbers in insets indicate the percentage of FLK1+
cells. (C') Results from three independent experiments are shown as a
percentage of BMP4 (D0) control. Error bars indicate s.e.m.
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Fig. 2. Gene expression analysis. (A) RNA from EBs differentiated either in serum
(FCS) or in serum-free conditions (SR), was subjected to semi-quantitative
RT-PCR. Numbers on top indicate days of EB differentiation. un,
undifferentiated ES cells. (B) EBs were generated in serum-free conditions
(SR) or SR+BMP4, collected at different time points, and RNA was subjected to
semi-quantitative RT-PCR. Numbers on top indicate days of differentiation. N,
negative H2O control.
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Fig. 3. BMP4 and VEGF are critical for the generation of SCL-expressing cells. (A)
Scl+/CD4 ES cells were in vitro differentiated in serum
(FCS) or serum-free conditions (SR) in the presence of factors shown. Day 5 EB
cells were subjected to FACS analysis for FLK1 and CD4 expression. Numbers in
a given box indicate the percentage of cells that are
FLK1+CD4– (lower right),
FLK1+CD4+ (upper right), or
FLK1–CD4+ (upper left). (B) Statistical analyses
of CD4+ cells developing in BMP4 (day 5-6 and 7) or BMP4+VEGF (day
5-6). (C) Both VEGF121 and VEGF165 are effective in
SCL-expressing cell induction. Day 7 Scl+/CD4 EB cells,
differentiated in serum-free conditions in the presence of BMP4,
BMP4+VEGF121, BMP4+VEGF165 or
BMP4+VEGF121+VEGF165 were subjected to FACS analysis for
FLK1 and CD4 expression. Numbers in a given box indicate the percentage of
cells that are FLK1+CD4– (lower right),
FLK1+CD4+ (upper right), or
FLK1–CD4+ (upper left).
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Fig. 4. Kinetic analyses of VEGF function. (A) Scl+/CD4 ES
cells were differentiated in serum-free conditions in the presence of BMP4 and
VEGF (upper panel) or BMP4 alone (lower panel). When ES cells were
differentiated in BMP4 and VEGF, the factors were removed on day 3 or 4 from
the medium by washing the EBs several times and returning them to fresh EB
differentiation medium without BMP4 and VEGF. When ES cells were
differentiated with BMP4, VEGF was added to the differentiation medium on day
3 or 4 of differentiation at 10 ng/ml. At day 5-6, the cells were subjected to
FACS analysis for FLK1 and CD4 expression. Numbers in a given box indicate the
percentage of cells that are FLK1+CD4– (lower
right), FLK1+CD4+ (upper right) or
FLK1–CD4+ (upper left). (B)
Scl+/CD4 ES cells were differentiated in serum-free
conditions in the presence of BMP4 and VEGF. The factors were washed off on
day 3 or day 4, and cells expressing Ter119 or CD31 were measured on day
6.
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Fig. 5. BMP4 induces phosphorylation of SMAD1/5, while VEGF induces phosphorylation
of Erk1/2 during EB differentiation. (A) ES cells were differentiated up to
day 1.5 (left) or day 2.75 (right) in serum-free conditions in the presence of
either bFGF or BMP4. (B) EBs harvested from day 1.5 differentiated in
serum-free conditions were treated with either bFGF or BMP4 for 30 and 60
minutes. (C) ES cells were differentiated in serum-free conditions with BMP4.
Three days after, BMP4 was washed off overnight and EBs were treated with VEGF
for 30 minutes. EB cell lysates were subjected to SDS-PAGE, followed by
immunoblotting with pSMAD1/5, SMAD1 (A,B), pERK1/2, ERK1/2, pAKT1/2/3 or
AKT1/2/3 (C). One out of three experiments is shown.
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Fig. 6. Overexpression of SMAD6 in ES cells inhibits the generation of
FLK1+ cells. (A) ES cells stably expressing SMAD6 were
differentiated and subjected to FACS analysis for FLK1 expression. Numbers in
insets indicate the percentage of FLK1+ cells. 2Ab indicates cells
stained with secondary antibody alone. Flag-6 and Flag-8 indicate control ES
cell clones. Flag SMAD6-6 and SMAD6-7 represent two independent ES cell clones
stably expressing Flag-SMAD6. Expression of exogenous Flag-SMAD6 was verified
with anti-FLAG antibody (arrow). (B) Results from four independent experiments
are shown as a percentage of Flag-6 control. Error bars indicate s.e.m.
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Fig. 7. The map kinase pathway is involved in the generation of SCL-expressing
cells. (A) ES cells were differentiated for three days in serum replacement
with BMP4. VEGF165 was added at day 3 with DMSO, U0124 or U0126
(MKK1-specific inhibitor). EBs were harvested at day 5 and subjected to FACS
analysis for FLK+ and SCL+ cell generation. Numbers in a
given box indicate the percentage of FLK1+CD4–
(lower right), FLK+CD4+ (upper right) and
FLK1–CD4+ (upper left) cells. (B) Results from
three independent experiments are shown as a percentage of U0124 control.
Error bars indicate s.e.m.
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Fig. 8. VEGF induces SCL-expressing cells mainly through the FLK1 receptor. (A)
Wild-type, Flk1–/– and
Flt1–/– ES clones were differentiated in serum
or serum-free conditions, in the presence of BMP4 and VEGF as shown. Day 5-6
EBs were analyzed for CD31 and TER119 expression. The percentage of TER119 and
CD31-positive cells are shown. One representative experiment out of three is
shown. (B) Flk1–/– and
Flt1–/– EB cells generated in the presence of
FCS (black bar), SR+BMP4 (white bar), or SR+BMP4+VEGF (grey bar) were
re-plated in a semi-solid medium as described in the Materials and methods.
Hematopoietic colonies were counted 5-7 days after re-plating. No
hematopoietic colonies formed when EB cells from SR were re-plated, thus not
shown. Error bars indicate standard deviations from triplicate plating. One
representative experiment out of three is shown.
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Fig. 9. TGFß1 and activin A in VEGF-mediated expansion of SCL-expressing
cells, hematopoietic and endothelial cell differentiation. Day 6
Scl+/CD4 EB cells generated in serum-free conditions in
the presence of BMP4; BMP4+VEGF; BMP4+VEGF+TGFß1; or BMP4+VEGF+activin A
were subjected to three-color analyses for FLK1, CD4 and hematopoietic or
endothelial markers. (A) The percentage of
FLK1+CD4– (lower right),
FLK1+CD4+ (upper right) or
FLK1–CD4+ (upper left) cells is shown. Statistical
values are shown on the right. (B) The percentage of TER119 and CD31-positive
cells is shown. (C) EB cells generated in the presence of FCS; SR+BMP4;
SR+BMP4+VEGF; SR+BMP4+VEGF+TGFß1; or SR+BMP4+VEGF+activin A were
subjected to hematopoietic re-plating. No hematopoietic colonies formed when
EB cells from SR were re-plated, thus not shown. Hematopoietic colonies were
counted 5-7 days after re-plating. Error bars indicate standard deviations
from triplicate plating.
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Fig. 10. A schematic diagram of BMP4, VEGF, activin A and TGFß1 action in
hematopoietic and endothelial cell development.
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© The Company of Biologists Ltd 2004
