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First published online 13 March 2008
doi: 10.1242/dev.011767
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Cancer Research UK, Paterson Institute for Cancer Research, Manchester University, Wilmslow Road, M20 4BX, Manchester, UK.
* Author for correspondence (e-mail: vkouskoff{at}picr.man.ac.uk)
Accepted 15 February 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Bmp4, Embryonic stem cell, Hemangioblast, Hematopoiesis, VEGF, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Palis et al.,
1999). This progressive differentiation can be monitored by
measurement of gene expression and analysis of biological potential by
secondary replating, which enables the quantification of all blood progenitors
via their colony-forming ability. As they lose their self-renewal and
pluripotent characteristics, ES cells form epiblast-like cells that
differentiate further to give rise to mesodermal precursors
(Kouskoff et al., 2005;
Pelton et al., 2002). The
first blood precursor, the hemangioblast, derives from the mesoderm and gives
rise to primitive and definitive hematopoiesis, smooth muscle and endothelium
(Ema et al., 2003;
Huber et al., 2004;
Yamashita et al., 2000). The
existence of this precursor has been established in vitro in mouse and human
ES differentiation models (Kennedy et al.,
2007; Lu et al.,
2007; Wang et al.,
2004), but also in vivo in mouse
(Huber et al., 2004) and
zebrafish (Vogeli et al.,
2006). However, Ueno and Weissman recently challenged the concept
of such a precursor through the direct clonal analysis of yolk sac blood
islands (Ueno and Weissman,
2006). The carefully orchestrated process that generates the
hemangioblast can be mimicked in vitro upon the differentiation of ES cells in
well-defined conditions; fetal calf serum being one of the key component of
these culture conditions (Choi et al.,
1998; Nishikawa et al.,
1998). Although the inherent characteristics of ES cell lines are
likely to be important for efficient differentiation, the batch of serum used
appears critical in obtaining the most effective differentiation. This fact
illustrates the complexity of serum composition and the many parameters that
may modulate, positively or negatively, the differentiation process. To better
dissect and understand the molecular mechanisms of cell fate specification,
refined culture conditions are needed to allow the specific and efficient
differentiation of ES cells toward hematopoiesis. Several serum-free culture
conditions have been described for the in vitro derivation of hematopoietic
cells from mouse ES cells (Adelman et al.,
2002; Ng et al.,
2005; Park et al.,
2004; Wiles and Johansson,
1997) or human ES cells (Tian
et al., 2004; Zambidis et al.,
2006). However, none of these previously published studies
described serum-free culture conditions that allowed a selective
differentiation of ES cells toward hematopoietic precursors, and that induced
the robust production of these progenitors. To optimize the culture conditions allowing the formation of blood progenitors upon ES cell differentiation in vitro, we tested several serum-free media and many growth factors. We found that only four factors were required for the very efficient and highly selective production of both primitive and definitive hematopoietic precursors. Our data show that the differentiation process can be separated into discrete steps that are each dependant on only one or two factors. Moreover, the stepwise nature of these culture conditions allowed us to further dissect the molecular program that regulates mesoderm specification at the onset of hematopoiesis.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Secondary colony assays
For the generation of blast cell colonies (BL-CFC assay), after
trypsinization (TrypLE Express, Gibco) EB cells were plated at a density of
2x104 cells/ml in 1% methylcellulose supplemented with 10%
FCS, VEGF (5 ng/ml), IL6 (5 ng/ml) and 25% D4T endothelial cell conditioned
medium (Choi et al., 1998). For
the growth of hematopoietic precursors, cells were plated at
2x104 cells/ml in 1% methylcellulose containing 10%
plasma-derived serum (PDS; Antech), 5% protein-free hybridoma medium (PFHM-II;
Gibco-BRL) and the following cytokines: KL (1% conditioned medium), TPO (5
ng/ml), erythropoietin (2 U/ml), IL11 (25 ng/ml), IL3 (1% conditioned medium),
GM-CSF (3 ng/ml), G-CSF (30 ng/ml), M-CSF (5 ng/ml) and IL6 (5 ng/ml).
Cultures were maintained at 37°C, 5% CO2. LIF, IL3 and c-Kit
ligand were derived from media as previously described
(Fehling et al., 2003). VEGF,
GM-CSF, M-CSF, G-CSF, TPO, IL6 and IL11 were purchased from R&D
Systems.
Matrigel plug assay
Individual vascular smooth muscle (VSM) or blast colonies were transferred
to 50 µl Matrigel plugs in 96-well plates. Matrigel (BD Biosciences) was
diluted 1:1 with IMDM supplemented with 10% FCS, 50 ng/ml VEGF, 5 ng/ml bFGF
and allowed to solidify at 37°C before the addition of colonies. Cultures
were maintained at 37°C, 5% CO2, 5% O2 for 4-8 days.
CD31 staining was performed on tubule-like structures after removal of most
Matrigel by vigorous washing in PBS (only possible for large structures).
After paraformaldehyde fixation for 30 minutes, tubule structures were
incubated in 10% serum in PBS for 30 minutes, followed by 4 hours incubation
with CD31-bio or non-specific CD3-bio control (BD Biosciences). After four
15-minute washes in PBS, bound antibody was revealed using Vectastain ABC-AP
and Blue-AP Substrate Kit III (Vector Laboratories) according to the
manufacturer's instructions.
Gene expression analysis
For gene-specific PCR, total RNA was extracted from each sample using the
RNeasy Plus Mini Kit (Qiagen). Two micrograms of RNA was reverse-transcribed
into cDNA using random hexamers and the Omniscript RT Kit (Qiagen). The PCR
reactions were performed using Biomix Taq (Bioline) and 0.2 µM each primer.
Cycling conditions were: 94°C for 5 minutes, followed by 30 cycles of
amplification (94°C denaturation for 30 seconds, 60°C annealing for 30
seconds, 72°C elongation for 60 seconds), then a final incubation at
72°C for 10 minutes. Sequences of the gene-specific PCR primers are
available upon request. Real-time PCR was performed on an ABI 7900 system
(Applied Biosystems) using Universal ProbeLibrary (Exiqon) and Primer Designer
(Roche). All expression data were calculated relative to actin controls as
2-
ct.
Flow cytometry
EBs were harvested, trypsinized, and the single-cell suspension was
directly analyzed for GFP expression or further stained. Cells were blocked
with FcR
II/III antibody (24G2 supernatant) prior to staining with
various combinations of antibodies CD45-FITC, Flk1-bio, CD41-PE, CD34-bio,
CD31-bio, Tie2-PE (all Pharmingen) or Ve-cad-647 (eBioscience) for 20 minutes
on ice in PBS containing 10% FCS, followed by strep-PECy5 or strep-PECy7
(Pharmingen) for 20 minutes on ice in PBS containing 10% FCS. Non-specific
antibody binding was always controlled for by staining cells with the
appropriate isotype controls. After two washes, cells were analyzed on a
FACSCalibur flow cytometer (BD Biosciences). For all analysis, flow cytometry
data were initially gated on the forward scatter versus side scatter profile,
enabling the separation of live cells from debris and dead cells.
Immunofluorescence
Single VSM colonies were picked, transferred on gelatin-coated coverslips
and cultured in IMDM supplemented with 10% FCS, 2 mM L-glutamine,
4.5x10-4 M MTG, VEGF (25 ng/ml), bFGF (20 ng/ml) and 25% D4T
endothelial cell conditioned medium. After 6-10 days, grown colonies were
fixed in 2% paraformaldehyde for 20 minutes, washed twice in PBS,
permeabilized in 0.2% Triton X-100 in PBS, washed in 10% FCS, 0.2% Tween 20 in
PBS, and then blocked with 10% FCS in PBS for 10 minutes. Colonies were
incubated for 1 hour with anti-CD31-bio and anti-smooth muscle actin-FITC
(Sigma). Bound anti-CD31 antibodies were visualized using a secondary
streptavidin-Cy3 (Caltag).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Chadwick et al.,
2003; Faloon et al.,
2000; Kennedy et al.,
2007; Park et al.,
2004; Wiles and Johansson,
1997). We used a mouse ES line (GFP-Bry) carrying the cDNA for
green fluorescent protein targeted to the brachyury locus to monitor
mesodermal differentiation (Fehling et
al., 2003). We have shown previously using this ES line that GFP
expression recapitulated effectively brachyury expression and could be used to
track mesoderm formation in vitro and in vivo
(Fehling et al., 2003;
Huber et al., 2004). Using
embryoid body (EB) formation to differentiate ES cells, we screened several
serum-free media as a base for the addition of growth factors, including
StemPro, Knockout Serum Replacement, IMDM/F12 and DMEM/F12 (all from Gibco).
Selection for a suitable serum-free basal medium was determined on the basis
of ES cell relative survival without induction of mesodermal differentiation
and without inhibition of differentiation upon serum addition. These criteria
were most effectively fulfilled with StemPro. Cell survival in StemPro medium
was comparable to that in serum-supplemented culture at day 1, but declined
progressively over the next 2 days of culture (see Fig. S1A in the
supplementary material). In contrast to serum-supplemented culture, no cell
expansion was observed (see Fig. S1A, right panel, in the supplementary
material) and no mesodermal differentiation was detected as illustrated by the
absence of GFP+ cells (Fig.
1A). Addition of serum to this serum-free medium at the start of
the EB culture induced the generation of mesodermal GFP-Bry+ cells
as well as the formation of committed blood progenitors co-expressing GFP-Bry
and VEGF receptor 2 (Flk1; also known as Kdr - Mouse Genome Informatics)
(Fig. 1A) as previously
described (Fehling et al.,
2003). Using this StemPro basal medium, we tested soluble factors
alone or in combinations according to the published literature
(Faloon et al., 2000;
Park et al., 2004;
Wiles and Johansson, 1997). A
mix of Bmp4, activin A, bFGF (Fgf2) and VEGF (VegfA) was best able to
recapitulate the effect of serum, although to a lesser extent, allowing the
generation of primitive and definitive hematopoietic precursors
(Fig. 1B). The relative
fraction of each type of definitive colony was not significantly different
whether EBs were grown in serum or with cytokine mixes (not shown).
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).
Analysis of EB-derived cells cultured for up to 3 days in serum-free
conditions without added factors showed that already by day 1, the expression
of Rex1 (Zfp42), a transcription factor specifically
expressed in ES cells, was strongly downregulated. In parallel, the expression
of Fgf5, which marks epiblast-like cells, was transiently upregulated
on day 1 and 2 (Fig. 1D).
Real-time PCR quantification confirmed that Rex1 was downregulated
5-fold by day 1 and more than 100-fold by day 3, whereas Fgf5 was
transiently upregulated up to 7-fold on day 1 and 2 (see Fig. S2 in the
supplementary material). The expression of both Oct4
(Pou5f1) and Nanog was maintained up to day 2 of
differentiation, but by day 3 the expression of these two genes was also
downregulated (Fig. 1D). These
changes in gene expression were accompanied by a strong decrease in the
potential to form EBs upon secondary replating when compared with the
potential of ES cells for generating EBs under similar conditions
(Fig. 1E). By the second day of
EB culture in serum-free media without added soluble factors, the transition
from ES cell to epiblast cell appeared complete: Rex1 expression was
barely detectable, Oct4 and Nanog were still expressed,
Fgf5 expression was strongly upregulated, and very few EBs were
generated upon secondary replating. The removal of leukemia inhibitor factor
(LIF) and feeder cells, which together keep ES cells undifferentiated, appears
sufficient to trigger the progression from ES cell to epiblast-like cell
stage. Analysis of the expression level of genes indicative of neuronal
(Sox1, Wnt1) or mesoderm/endoderm (Gata4, brachyury,
Sox17) differentiation, did not reveal any overt sign of random
differentiation toward any of these lineages
(Fig. 1D). Our data indicate
that no exogenously added factors are necessary for the transition from ES
cells to epiblast-like cells. Although ES cells were able to progress to
epiblast-like cells, we never detected GFP expression driven from the
brachyury locus.
Bmp4 promotes the efficient formation of mesoderm but a poor specification to hemangioblast fate
The next step of differentiation, the progression from epiblast-like cells
to mesoderm, was monitored by assessing the percentage of GFP+
cells during a time-course of GFP-Bry ES cell differentiation. Various
cytokines were tested for their potential to induce mesoderm when added at the
start of the EB culture. Bmp4 induced robust mesoderm commitment, whereas VEFG
and bFGF did not induce significant GFP expression, and activin A induced only
limited GFP upregulation (Fig.
2A,C and see Fig. S3 in the supplementary material). When added at
4 ng/ml, Bmp4 induced a pattern of GFP+ cell formation highly
similar to that seen in the serum condition. By day 2.5 of differentiation,
the first GFP+ cells were detected. This percentage of
GFP+ cells increased steadily to reach nearly 100% by day 3.75,
when the cells started to downregulate GFP expression, as previously observed
(Fehling et al., 2003). A dose
titration of Bmp4 showed that although a wide range of concentrations induced
GFP+ cell formation, both low (0.8 ng/ml) and high (100 ng/ml)
concentrations were suboptimal for mesoderm induction
(Fig. 2A). The level of GFP
expression was not influenced by the Bmp4 concentration (see Fig. S4A in the
supplementary material). Interestingly, at day 4 of differentiation, all Bmp4
concentrations tested led to a slight increase in GFP level relative to the
serum control. A dramatic increase in GFP+ cell recovery was
observed between day 2 and 3 of differentiation, which correlates with the
onset of mesoderm formation and supports the inductive rather than survival
nature of Bmp4 action (see Fig. S4B in the supplementary material). In support
of a lack of direct correlation between survival and induction, Bcl2
overexpression in ES cells led to a 5-fold increase in survival upon EB
formation in serum-free conditions, but this enhanced survival did not replace
the requirement for Bmp4 to promote mesoderm specification (see Fig. S1B,C in
the supplementary material). In addition to brachyury upregulation, Bmp4
stimulation induced the upregulation of several genes indicative of paraxial
mesoderm, including Fgf8, follistatin, Mesp2 and
Tbx6 (Fig. 2B). These
data suggest that Bmp4 might be able to stimulate the formation of a broad
range of mesoderm subpopulations. Our data do not fully address the direct
action of Bmp4 on epiblast-like cells. However, it is unlikely that Bmp4 acts
through an intermediate visceral endoderm-like layer. Indeed, we failed to
detect the presence of a visceral endoderm-like layer in EBs, either by
morphology or by DBA (Dolichos biflorus agglutinin) staining, whereas
this layer was clearly stained with DBA in E7.25 embryos (see Fig. S5 in the
supplementary material).
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). These
hemangioblast precursors can be followed by the upregulation of VEGF receptor
2 (Flk1) and by their ability to form blast colonies in secondary replating
assays (Choi et al., 1998;
Faloon et al., 2000). These
blast colonies are able to generate hematopoiesis, endothelium and smooth
muscle lineages when further cultured under appropriate conditions
(Ema et al., 2003;
Huber et al., 2004;
Yamashita et al., 2000). Upon
stimulation with Bmp4, we observed the upregulation of Flk1 expression in
around 30% of EB-derived cells (Fig.
2C). However, despite Flk1 expression, very few blast colonies
were generated in secondary replating (Fig.
2D); this was observed at all Bmp4 concentrations tested (not
shown) and the main type of colonies generated had a smooth and compact aspect
(Fig. 2F, top panel). Staining
of pooled colonies revealed the presence of a Tie2+CD45-
subpopulation (Fig. 2E) that
also expressed VE-cadherin (cadherin 5) and CD31 (Pecam1) at low level (not
shown) and is likely to represent endothelial cells. These colonies gave rise
upon further culture to endothelial and smooth muscle cells as defined by cell
morphology and by their respective expression of CD31 and smooth muscle actin
(Fig. 2F, lower panel). When
cultured in Matrigel plugs, individual colonies gave rise to a tubule-like
structure, further indication of their endothelial nature
(Fig. 2G). These colonies,
termed vascular smooth muscle (VSM) colonies, have been described previously
(D'Souza et al., 2005).
Overall, these data demonstrate that in a dose-dependant manner, Bmp4 is able
to induce the very efficient formation of mesodermal precursors, as nearly
100% of the cells become GFP+. Furthermore, Bmp4 stimulation
results in Flk1 upregulation, but Bmp4 alone is not sufficient to promote the
robust generation of hemangioblast precursors. The main type of precursors
generated in the presence of Bmp4 appears to be restricted to the endothelium
and smooth muscle fate.
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; Huber et al.,
2004). Upon further differentiation, they will give rise to
restricted precursors for both primitive and definitive hematopoietic lineages
(Choi et al., 1998;
Yamashita et al., 2000). The
formation of these more-committed blood precursors upon sequential induction
by Bmp4, activin A and bFGF, was assessed at later time points by secondary
replating allowing the formation of primitive and definitive colonies. As
shown in Fig. 3F, when compared
with the serum condition at all time points analyzed, few primitive or
definitive colonies were derived from EBs grown in serum-free media
supplemented either with Bmp4 alone or with Bmp4, activin A and bFGF.
Altogether, these data show that mesodermal precursors were able to
efficiently and rapidly form hemangioblast upon stimulation with bFGF and
activin A. However, although hemangioblast formation was very robust, these
hemangioblasts failed to further commit to more-mature hematopoietic
precursors without the addition of growth factors other than Bmp4, activin A
and bFGF in the serum-free culture conditions.
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IIb integrin subunit (CD41; Itga2b) has been shown to define the onset
of primitive and definitive hematopoiesis both in vivo and in vitro
(Emambokus and Frampton, 2003;
Ferkowicz et al., 2003;
Mikkola et al., 2003;
Mitjavila-Garcia et al.,
2002). By day 6 of differentiation, most cells within the
BAFV-stimulated EBs belonged to the hematopoietic lineage as defined by their
expression of CD41 (Fig. 4B).
At days 5 and 6, few cells within the BAFV-stimulated EBs expressed markers of
more-mature hematopoiesis such as CD45 (Ptprc) and Mac1 (Itgam), but by day 7
CD45+Mac1- and CD45+Mac1+
subpopulations were clearly detected (Fig.
4C). All hematopoietic progenitors were found in the
CD41+ fractions (Fig.
5A), both primitive and definitive precursors were present in the
CD41+CD34- subpopulation, and
CD41+CD34+ potential was restricted to definitive
progenitors. The main population, CD41+CD34-, was mostly
composed of immature blastic cells with a few more-mature cells but no mature
megakaryocytes (Fig. 5B).
Attempts to generate B or T lymphocytes on OP9 or OP9-Dll1 stroma cell lines
were unsuccessful for all fractions (not shown). To further characterize the
CD41+ population, we analyzed Runx1 expression within this
population using an ES cell line in which the truncated human CD4 has been
knocked into the Runx1 locus (P.S., V.K. and G.L., unpublished).
Runx1 expression was detected in most CD41+ cells
(Fig. 5C), another indication
that most CD41+ cells are committed to the hematopoietic program.
In Bmp4-stimulated EBs, the main positively stained population was
CD34+CD41-. When tested in replating assays after
purification, this population only gave rise to VSM endothelium colonies
(Fig. 5A,D). Upon expansion in
three-dimensional Matrigel plugs, these VSM colonies gave rise to tubule-like
structures (not shown). Altogether, these data demonstrate that VEGF is
sufficient to trigger the commitment and/or expansion of more-mature blood
precursors for both primitive and definitive lineages. It is very unlikely
that VEGF acts as a mere survival factor, as Bcl2 overexpression was not able
to substitute for VEGF for the efficient production of hematopoietic
precursors (not shown). The very high proportion of CD41+
hematopoietic cells within the EBs further highlights the efficiency with
which this combination of factors specifically promotes hematopoietic
commitment at the expense of other lineages.
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Analysis of the molecular program at the onset of hemangioblast specification
The serum-free culture conditions described above represent a perfect
system with which to further dissect the molecular program established at the
onset of blood specification. To analyze the expression pattern of genes
indicative of the differentiation process, we first performed an expression
survey over the time-course of the EB culture from day 0 to 6
(Fig. 6A), with Bmp4 added at
time 0 only (lanes B), or with Bmp4 followed by activin A and bFGF addition at
day 2.5 (lanes BAF) or by activin A, bFGF and VEGF addition at day 2.5 (lanes
BAFV). By day 1 of differentiation, Rex1 expression was markedly
decreased, followed by the progressive upregulation of Fgf5,
indicating a transition from ES cells to epiblast-like cells. Expression of
brachyury was detected after 2 days, a mark of commitment to the mesodermal
germ layer. Interestingly, brachyury expression seemed to be downregulated
faster when cells were stimulated with BAF or BAFV than with Bmp4 only.
Flk1 expression was detected by day 3 in all conditions and was
shortly followed by the expression of genes implicated in hematopoietic
commitment such as Scl (Tal1), Runx1 and
Gata1, and by βH1, the embryonic hemoglobin expressed only in
primitive erythrocytes. All genes indicative of hematopoietic commitment were
expressed at a low level in EBs stimulated with Bmp4 only or under BAF
conditions. However, the expression of this set of genes was strongly
upregulated when VEGF was added at day 2.5. This expression study nicely
complements the hematopoietic potential data
(Fig. 3D,
Fig. 4A) showing that few
precursors were generated within EBs in the absence of VEGF induction. To
further refine our study of the molecular program that is established at the
onset of hematopoiesis commitment, we analyzed gene expression following the
stimulation of mesodermal precursors by activin A and bFGF. After 2.5 days of
culture with Bmp4, EBs were stimulated with activin A and bFGF and harvested
after 1, 2, 3 or 6 hours of induction to assess the dynamic expression of some
genes implicated as being crucial in hematopoietic development
(Fig. 6B,C). Within 2 hours of
induction, the expression of Runx1 was significantly upregulated, and
increased expression of Hhex was detected 3 hours following
induction. Expression of Scl, Fli1 and Lmo2 became
significantly upregulated by 6 hours, although a small increase in
Scl expression level could already be observed 1 hour after induction
(Fig. 6B). By contrast, we did
not observe any significant upregulation of Gata1, Gata2, cMyb or
Pu.1 (Sfpi1) (Fig.
6C): Gata2 and Pu.1 were expressed at a low and
steady level, whereas Gata1 and cMyb were below detection
level - expression of these genes was successfully detected using cDNA derived
from day 4.5 EBs grown with serum (Fig.
6C, the C+ panel).
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The strongly upregulated expression of known transcriptional regulators of hematopoiesis associated with a robust increase in the number of hemangioblast precursors can be detected within a few hours of stimulation with activin A and bFGF (Fig. 3B, Fig. 6B). This very rapid response suggests that activin A and bFGF act together to promote the specification of mesodermal precursors to the hematopoietic program, rather than inducing the proliferation of a few hemangioblasts already present in the EBs. The strongest argument in favor of induction rather than proliferation relates to the kinetics of hemangioblast generation. Three hours after activin A and bFGF addition, we observe an average 8-fold increase in hemangioblast numbers (Fig. 3B). This increase cannot be exclusively the consequence of proliferation, as not even the fastest proliferating eukaryotic cell can divide two or three times in 3 hours. Furthermore, the hemangioblast assay is a clonal assay in semi-solid media and, consequently, if proliferation occurred after the replating this would still result in the formation and detection of only one blast colony.
Our results suggest that VEGF is not critically required for the formation
of hemangioblast, but is essential for the formation and/or proliferation of
both primitive and definitive hematopoietic precursors. In the replating
assay, the formation of blast colonies requires the action of VEGF. However,
the formation of blast colonies represents the further development of
hematopoietic and endothelium progenitors from an already formed
hemangioblast. Hemangioblast precursors express Flk1, but this does not imply
that they require signaling through Flk1 or another VEGF receptor for their
generation. To date, no published findings demonstrate the requirement for
VEGF in the formation of hemangioblast precursors. In fact, data from Ema et
al. suggest that without Flk1, hemangioblast and blast colonies are still
generated, although to a lesser extent than in wild-type controls
(Ema et al., 2003).
Our data demonstrate that Bmp4 alone, or a combination of Bmp4, activin A
and bFGF, is able to stimulate the formation of some fully committed blood
progenitors, but that the addition of VEGF dramatically enhances the frequency
of these precursors (Fig. 4A).
There are no statistical differences in the production of committed
hematopoietic precursors (excluding the hemangioblast precursors) when ES
cells are differentiated with Bmp4 alone or with Bmp4, activin A and bFGF
(Fig. 3F,
Fig. 4A). By contrast, the
addition of VEGF resulted on average in a 250-fold increase for primitive and
a 170-fold increase for definitive colonies when compared with Bmp4 induction
alone (Fig. 4A). Even when
compared with serum conditions, VEGF addition resulted in 3-fold and 6-fold
increases for primitive and definitive colonies, respectively. Overall, the
frequency of hematopoietic colonies generated by sequential stimulation by
Bmp4 followed by activin A, bFGF and VEGF is higher than any other culture
conditions tested in the present study. Two recent studies have explored the
growth factor requirements for the derivation of hematopoietic precursors from
human ES cells (Kennedy et al.,
2007; Pick et al.,
2007). Kennedy et al. showed that sequential addition of BMP4,
followed by bFGF then by bFGF plus VEGF in StemPro medium triggered the
formation of hemangioblast and more-mature hematopoietic precursors. In the
second study, Pick et al. defined the combination of BMP4, VEGF, SCF (KITLG -
Human Gene Nomenclature Database) and bFGF as being required to promote
hematopoiesis as assessed by gene expression and colony-forming cell (CFC)
formation. In both studies, however, the production of hematopoiesis was
clearly not as efficient as in our culture conditions, and these studies did
not address the precise role of each added factor. It is difficult, however,
to directly compare the efficiency of culture conditions for human ES and
mouse ES cells. In our culture conditions, as well as in these two human ES
cell studies, Bmp4, bFGF and VEGF appear to be crucial for hematopoiesis. It
would be very interesting to test whether the addition of activin A to EBs
derived from human ES cells enhances the efficiency of progenitor
generation.
The crucial role of Bmp4 during mesoderm development in vivo has already
been clearly established using various knockout mice
(Fujiwara et al., 2001;
Gu et al., 1999;
Mishina et al., 1995;
Winnier et al., 1995). VEGF
and its receptor Flk1 have also been shown to be crucial at the onset of blood
and vasculature development during embryogenesis
(Carmeliet et al., 1996;
Ferrara et al., 1996;
Shalaby et al., 1995). By
contrast, a role for activin A during hematopoietic development in vivo seems
unlikely as activin A-/- mice have no reported blood defects
(Matzuk et al., 1995).
However, mice deficient for TGFβ1, a closely related member of TGF
family, were shown to have vasculature and hematopoietic defects during
development (Dickson et al.,
1995). When assayed in the serum-free culture system described
here, we observed that similarly to activin A, TGFβ1 in conjunction with
bFGF was able to induce the very robust formation of hemangioblast (not
shown).
These culture conditions represent a powerful system with which to dissect
the molecular mechanisms implicated in hemangioblast commitment. The
possibility to switch on the hematopoietic program with activin A and bFGF
allowed us to perform a time-course of gene expression dynamics at the onset
of hemangioblast commitment and to pinpoint genes that are crucial regulators
of this commitment. Runx1, Scl and Hhex, which have been shown to regulate
hemangioblast development (Chung et al.,
2002; D'Souza et al.,
2005; Ema and Rossant,
2003; Guo et al.,
2003; Kubo et al.,
2005; Lacaud et al.,
2002), were progressively upregulated upon activin A plus bFGF
induction. Six hours post-induction, increased expression was also observed
for Fli1 and Lmo2, two transcription factors known to be important in the
development of the hematopoietic program
(Hart et al., 2000;
Spyropoulos et al., 2000;
Warren et al., 1994). Recent
findings in zebrafish have highlighted the crucial role of Lmo2 in
hemangioblast specification (Patterson et
al., 2007), whereas a direct role for Fli1 in this process remains
to be demonstrated. By contrast, we did not observe any significant changes in
either Gata1 or Gata2 expression upon hemangioblast specification, despite
reports of their involvement in this process
(Lugus et al., 2007;
Yokomizo et al., 2007). Gata2
function in hemangioblast development was assessed via an inducible
overexpression. Rather than resulting from a direct effect of Gata2, increase
in hemangioblast generation might have resulted from Gata2 expression
impacting on Scl expression (Lugus et al.,
2007). Gata2 is possibly not directly implicated in the
establishment of the hemangioblast program, but its expression is essential
for Scl expression (Gottgens et al.,
2002). In our study, Gata1 expression was clearly not detected
upon activin A plus bFGF induction, even after 24 hours
(Fig. 6C). However, Yokomizo et
al. have shown using a GFP mini-transgene that hemangioblastic cells are
positive for Gata1 expression (Yokomizo et
al., 2007). A possible explanation for this discrepancy is that
the mini-transgene used to mimic Gata1 expression did not fully recapitulate
the Gata1 expression pattern and that negative regulatory regions might be
missing. Overall, our gene expression survey further highlights the key role
of the Runx1, Scl, Hhex and Lmo2 transcription factors in regulating the onset
of hematopoietic development. Importantly, these data underscore the power of
our serum-free culture conditions, which allow us to catch mesodermal
precursors as they become committed to hematopoiesis, and potentially to
define new molecular players implicated in this process.
The true contribution of hemangioblast precursors to yolk sac hematopoiesis
has been recently challenged (Ueno and
Weissman, 2006). The apparent polyclonal origin of the blood
island of yolk sac observed in this study might in fact have resulted from the
very transient nature of this precursor. Indeed, hemangioblast progenitors are
mostly detected in the primitive streak at around 7.5 days post-coitum
(Huber et al., 2004), and will
have already progressed in their differentiation toward hematopoietic- and/or
endothelium-restricted precursors when reaching the yolk sac, within which
they are very likely to intermingle at this point.
In the present study, we have defined robust and reproducible culture conditions to specifically and efficiently drive hematopoietic differentiation from ES cells in the absence of serum. This system should help us to better dissect and understand early lineage commitment to hematopoiesis.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/8/1525/DC1
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ACKNOWLEDGMENTS |
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