MRC Centre Development in Stem Cell Biology, Institute for Stem Cell Research, University of Edinburgh, King's Buildings, West Mains Road, Edinburgh EH9 3JQ, UK
Author for correspondence (e-mail:
a.medvinsky{at}ed.ac.uk)
Accepted 8 July 2005
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: AGM region, Yolk sac, Stem cells, VE-cadherin, Mouse
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A large body of data suggests that endothelial and haematopoietic cells
have a common origin in development. Endothelial and haematopoietic cells
share a number of common markers (Godin
and Cumano, 2002). Experiments using ES cell differentiation
models have shown that adult-type clonogenic haematopoietic progenitors can
originate from the endothelial VE-cadherin+CD45- cells
(Fujimoto et al., 2001
). The
relationship between endothelial and haematopoietic cells has also been
clonally explored using an ES cell blast colony assay based on FLK1 and
PECAM/CD31 expression (Chung et al.,
2002
; Ema and Rossant,
2003
; Fehling et al.,
2003
; Kennedy et al.,
1997
; Lacaud et al.,
2002
).
FLK1+ progenitor cells committed to endothelio-haematopoietic
differentiation have recently been localised to the primitive streak of the
E7.0-7.5 mouse embryo (Huber et al.,
2004). In the more advanced embryo, VE-cadherin+ cells
are also a source of haematopoietic cells
(Nishikawa et al., 1998a
).
Immunohistochemical analysis suggests that some cells of the embryonic dorsal
aorta co-express endothelial-specific VE-cadherin and haematopoietic markers
(Breier et al., 1996
;
Carmeliet et al., 1999
;
Fraser et al., 2003
). Clusters
of haematopoietic cells adhered to the endothelium of the dorsal aorta
(Jordan, 1917
;
Medvinsky et al., 1996
;
North et al., 1999
;
Tavian et al., 1996
) and the
umbilical cord (North et al.,
1999
) are often interpreted as haematopoietic cells budding off
from the endothelial lining. Occasional disruption of the endothelial basal
membrane underlying such clusters suggest active involvement of local
endothelium (Tavian et al.,
1999
). Labelling of chick embryonic endothelium in ovo resulted in
the subsequent appearance of labelled haematopoietic cells, consistent with an
endothelial origin of haematopoiesis
(Jaffredo et al., 2000
;
Jaffredo et al., 1998
). Recent
data have suggested the origin of HSCs from sub-endothelial patches/mesenchyme
(Bertrand et al., 2005
;
North et al., 2002
). However,
the relation of these cells to the endothelial lineage has yet to be unveiled.
There is also some experimental evidence suggesting the existence of the
haemangioblast in the adult bone marrow
(Bailey and Fleming, 2003
;
Pelosi et al., 2002
).
Furthermore, key regulators of vasculo- and angiogenesis, VEGF and
angiopoietin 1, play crucial roles in the maintenance of HSCs in adult bone
marrow (Gerber et al., 2002
;
Takakura et al., 1998
).
Angiopoietin signalling may be involved in embryonic development of HSCs
(Hsu et al., 2000
;
Yuasa et al., 2002
). Although
some controversy remains, a lineage relationship between haematopoietic and
endothelial differentiation is currently widely accepted.
The definitive haematopoietic hierarchy develops from definitive HSCs
(Kondo et al., 2003), the
appearance of which in the embryo follows a complex developmental pattern
(de Bruijn et al., 2000
;
Gekas et al., 2005
;
Kumaravelu et al., 2002
;
Medvinsky et al., 1996
;
Muller et al., 1994
;
Ottersbach and Dzierzak,
2005
). By late E10.5-E11.5, the first definitive HSCs appear in
the AGM region, the umbilical vessels and then slightly later in the YS.
Recently, the E10.5-E13 placenta has also been identified as an early abundant
reservoir of HSCs (Gekas et al.,
2005
; Ottersbach and Dzierzak,
2005
). Of note, high level repopulating activity (>5%) appears
only from E11 (Kumaravelu et al.,
2002
; Medvinsky and Dzierzak,
1996
). The growing number of definitive HSCs in embryonic blood
from E11.5 correlates with rapid liver colonization
(Christensen et al., 2004
;
Ema and Nakauchi, 2000
;
Kumaravelu et al., 2002
;
Morrison et al., 1995
). As
shown by the organ culture approach, the AGM region by E11.5 acquires the
capacity to initiate/expand HSCs but by E12.5 HSC generation is overtaken by
the YS (Kumaravelu et al.,
2002
; Medvinsky and Dzierzak,
1996
), which supports the suggestion that the YS also contributes
to definitive haematopoiesis (Toles et
al., 1989
; Weissman et al.,
1978
; Yoder et al.,
1997
). The capacity of the placenta to expand HSCs is an important
issue that has yet to be investigated. Collectively, these data show a
dramatic increase in HSC numbers between E11.5 and E12.5 in the entire
conceptus, including the foetal liver and the placenta, implying a massive
initiation of new HSCs in the embryo
(Gekas et al., 2005
;
Kumaravelu et al., 2002
).
Here, using in vivo and in vitro techniques (Fig. 1), we have explored the evolution of the endothelial character of definitive HSCs during their initiation, migration and hepatic colonization. At initiation, HSCs reside exclusively within a population defined by the co-expression of both endothelial and haematopoietic markers. The HSCs largely retain endothelial markers during circulation until they colonise the liver where VE-cadherin is downregulated. However, the developmental switch of HSCs from the VE-cadherin positive to negative phenotype does not require contact with the liver.
|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissue preparation and organ culture
E11.5 AGM, E12.5 YS and E13.5 FL were dissected from embryos. Special care
was taken to remove large vessels from the YS. Peripheral blood (PB) was
collected from E12.5 embryos as previously described
(Kumaravelu et al., 2002).
Dissected organs were incubated in 0.1% collagenase-dispase (Roche)/PBS
(Sigma) at 37°C for 40 minutes and then dissociated by gentle manual
pipetting. Prior to antibody labelling PB and FL suspensions were depleted of
erythrocytes using anti-Ter119 antibody conjugated magnetic microbeads
(Miltenyi Biotec). Organ cultures were set up as described previously
(Kumaravelu et al., 2002
).
E11.5 and E12.5 placenta were prepared as previously described
(Gekas et al., 2005
).
Flow cytometry
Monoclonal antibodies against the following antigens were used: AA4.1-APC,
4-integrin-PE, B220-biotin, CD3
-PE, CD4-PE, CD8
-Biotin,
CD34-FITC/PE, CD41-FITC, CD45-PE/FITC, KIT (also known as C-KIT)-PE/APC,
FLK1-PE, GR1-Bbotin, Ly5.1-PE, Ly5.2-FITC, MAC1-PE, SCA1-PE, TIE2-PE,
VE-cadherin-biotin and CD16/32-purified. Biotin was detected using APC- or
PE-conjugated streptavidin. Appropriate isotype controls were used. Dead cells
were excluded using 7-AAD. Reagents were purchased from eBioscience and
Pharmingen.
MoFlo (DakoCytomation) and FACStar (Beckton-Dickinson) flow cytometers were used for sorting. A FACScalibur (Beckton-Dickinson) was used for flow cytometric analysis. Data analysis was performed using FlowJo software (TreeStar).
Long-term repopulation assay
Competitive transplantation experiments were set up as previously described
(Kumaravelu et al., 2002). The
transplanted embryonic cells are expressed throughout the paper in embryo
equivalents (e.e.), defined as a unit of (sorted) cells equivalent to the
number of cells of that phenotype contained in one organ. The transplanted
bone marrow cells are expressed in HSC equivalents (HSC.e.): one HSC.e. is
equivalent to 10,000 nucleated bone marrow cells, which harbours 1 HSC
(Kumaravelu et al., 2002
).
Cytological examination
Following cell sorting, E11.5 AGM fractions were centrifuged at 1000 rpm
for 4 minutes (Cytospin 3, Shandon) onto poly-L-lysine-coated slides (BDH).
Preparations were fixed in methanol for 2.5 minutes and stained with
May-Grunwald and Giemsa stains (BDH). Images were taken with an Axiovert S100
microscope (Zeiss) using Openlab software (Improvison). Images were prepared
using Adobe Photoshop.
Clonogenic myeloid progenitor assay
Sorted cells from E11.5 AGM were cultured in methylcellulose medium (M3434,
Stem Cell Technologies) according to the manufacturer's instructions.
Haematopoietic colonies were scored between 10 and 12 days.
|
Single-cell progenitor assay
OP9 haematopoietic differentiation assay was performed as described
previously (Nishikawa et al.,
1998b) with slight modifications. Briefly, confluent OP9 layers in
96-well plates were seeded with flow cytometrically sorted cells and incubated
in
-MEM medium supplemented with FCS (10%), EPO (1U/ml), IL3 (200U/ml),
Pokeweed mitogen spleen-conditioned medium (2%) and G-CSF-conditioned medium
(1%). Following 7 days of culture, the wells containing round cells on top of
the OP9 layer were counted. The haematopoietic identity of these cells was
confirmed by flow cytometric analysis following CD45 antibody staining.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
To understand more precisely the identity of the DP fraction markers
associated with early haematopoietic differentiation were also examined.
4-Integrin labels a large number of adult haematopoietic cells
(Arroyo et al., 1996
) and marks
haematogenic endothelial activity (Ogawa
et al., 1999
). In the E11.5 AGM region
4-integrin
expression marks
3% of endothelial cells but in the DP and haematopoietic
populations virtually all cells express
4-integrin
(Fig. 2B). Finally, the
expression of CD41, a traditional megakaryocyte/platelet marker implicated in
early haematopoietic development was also analysed
(Bertrand et al., 2005
;
Emambokus and Frampton, 2003
;
Ferkowicz et al., 2003
;
Mikkola et al., 2003
;
Mitjavila-Garcia et al.,
2002
). A fraction of the DP population was found to contain
CD41-positive cells.
Thus, the DP population is uniquely promiscuous. Not only does this fraction co-express the predominantly mutually exclusive VE-cadherin and CD45 determinants, but it also bears cardinal markers of endothelial and haematopoietic/stem cell differentiation.
E11.5 AGM clonogenic myeloid progenitors are enriched in the DP fraction
We next investigated the morphology of the E11.5 AGM endothelial
(VE-cadherin+CD45-), haematopoietic
(VE-cadherin-CD45+) and DP
(VE-cadherin+CD45+) populations. Cytospin preparations
from sorted fractions revealed that the endothelial fraction contained mainly
cells with large patchy stained nuclei and blebbing cytoplasm
(Fig. 3). Vacuolated cytoplasm
of endothelial cells outlining the embryonic dorsal aorta was noticed
previously on histological sections (North
et al., 1999). The majority of DP cells demonstrated a high
nuclear-to-cytoplasmic ratio, characteristic for blast/stem cells. The
haematopoietic population consisted mainly of more mature cells with large,
often vacuolated, cytoplasm and small nuclei of variable shape.
|
We then investigated whether co-culture with the OP9 stromal cell line would unveil haematopoietic potential in non-DP cells. In limiting dilution experiments, we found that clonogenic progenitors in the E11.5 AGM region resided exclusively within the DP population, at an average frequency 7.6% (Fig. 4D-F). Other cell fractions failed to produce any detectable numbers of haematopoietic cells.
Endothelial network formation is restricted to the VE-cadherin+CD45- population
To investigate the possibility that the DP population might encompass the
in vivo haemangioblast, we tested the in vitro endothelial
differentiation/network forming capacity of the four E11.5 AGM fractions using
a well established assay (Nishikawa et
al., 1998b). We found that endothelial tubule and network
formation was largely restricted to the endothelial
(VE-cadherin+CD45-) population: 500 endothelial cells
formed about four PECAM1+ tubules, while 5000 endothelial cells
formed
60 tubules and 20,000 endothelial cells resulted in extensive
network formation (Fig. 4G-I).
By contrast, only two tubules were formed with 50,000
VE-cadherin-CD45- plated cells and no endothelial
capacity was observed within the haematopoietic or DP populations
(Fig. 4G). Therefore, despite
clear phenotypic similarity with the endothelium, the DP population has
functionally diverged from the endothelial compartment.
Definitive HSCs in the E11.5 AGM region reside within the DP cell fraction
E11.5 AGM region was flow sorted on the basis of VE-cadherin and CD45
expression. In line with previous reports
(North et al., 2002) all HSC
activity was detected within the VE-cadherin+CD45+
population. From a cohort of 12 adult recipients, four were repopulated with
the DP population, with a range of 5-60% peripheral blood leukocyte chimerism
(average of 36%) (Table 2). As
one E11.5 AGM region harbours 1 HSC
(Kumaravelu et al., 2002
), the
frequency of HSCs in the DP population is estimated at 1 in 70 cells
(Table 3).
|
|
It has been shown that VE-cadherin+ cells from the E9.5 YS are
restricted in their capacity for effective myeloid differentiation following
engraftment in neonatal animals (Fraser et
al., 2002). On occasions, we also observed a bias towards lymphoid
contribution from E11.5 AGM and E12.5 PB HSCs. Therefore, as long-lived
lymphoid cells can circulate in the absence of HSCs, we performed a more
detailed analysis. We found long-term production of short-lived donor-derived
MAC1+GR1+ granulocytes especially in bone marrow
(Fig. 5A). We also demonstrated
donor-derived CD43+B220+ pre/pro-B-cells in bone marrow
and CD4+CD8+ cells in the thymus, demonstrating
long-term contribution of donor HSCs. Furthermore, successful secondary
transplantation demonstrates contribution into the stem cell compartment of
the primary recipient (Fig.
5B).
|
At the peak of HSC activity in the YS at E12.5
(Kumaravelu et al., 2002),
sorted VE-cadherin+ and VE-cadherin- cell populations
were transplanted into irradiated adults. Out of 10 recipients that received
transplants of VE-cadherin+ cells, seven mice were repopulated at
the range of 5-87% with an average of 33%
(Table 2). No reconstituting
capacity was detected within the VE-cadherin- fraction.
CD45+ and CD45- fractions from E12.5 YS were also
separately transplanted. Three out of three mice were repopulated with
CD45+ cells (61-87% range of chimerism with an average of 74%),
whereas one out of four recipients was repopulated with the CD45-
fraction, possibly reflecting contamination with CD45+ cells.
We conclude that as in the E11.5 AGM all definitive HSCs in the E12.5 YS
reside within the DP population. As the E12.5 YS contains about two HSCs
(Kumaravelu et al., 2002), the
DP population in this tissue is highly enriched for HSC activity (1/64 cells)
(Table 3). Thus, HSCs emerging
both in the E11.5 AGM and E12.5 YS co-express VE-cadherin and CD45.
Downregulation of VE-cadherin in E12.5 AGM HSCs
By E12.5 the HSC activity in the AGM region decreases
(Kumaravelu et al., 2002). At
this more advanced stage (in contrast to E11.5), the AGM region contains some
VE-cadherin- cells that are capable of repopulating
(Table 2): all HSCs in E12.5
AGM region however remain CD45+. These data suggest that VE-cadherin is
downregulated as HSC development progresses.
VE-cadherin phenotype of placental CD34+KIT+ fraction
Recent reports have shown that HSCs in the placenta reside within
CD34+KIT+ fraction
(Gekas et al., 2005;
Ottersbach and Dzierzak,
2005
). We therefore analysed VE-cadherin and CD45 expression in
this placental population (Fig.
7A-C). At E11.5 the entire CD34+KIT+
population was VE-cadherin+CD45+
(Fig. 7B). At E12.5, the
CD34+KIT+ population retains CD45+ but only
30±3.0% remain VE-cadherin+
(Fig. 7C), indicating that, as
in the AGM region, downregulation of VE-cadherin at the phenotypic level may
occur in the placental HSC fraction.
Most definitive HSCs in the E13.5 foetal liver and all HSCs in the adult bone marrow are VE-cadherin negative
HSCs born in extra hepatic sites colonise the foetal liver via the
circulation. Therefore we investigated the phenotype of HSCs present in the
E13.5 foetal liver. Following the purification of
VE-cadherin+CD45+ and
VE-cadherin-CD45+ fractions each recipient received 0.02
embryo equivalent (e.e.) of foetal liver cells, equating to approximately five
stem cells (Kumaravelu et al.,
2002) (Table 2).
While all five recipients of the haematopoietic fraction were repopulated,
only four out of five mice transplanted with the DP fraction were successfully
reconstituted. No obvious difference was observed in multi-lineage
differentiation capacity between VE-cadherin+CD45+ and
VE-cadherin-CD45+ HSCs isolated from the E13.5 liver
(data not shown). We also found that both VE-cadherin+ and
VE-cadherin- HSCs were capable of repopulating secondary recipients
(Fig. 5B).
It is difficult to assess accurately the actual numbers of HSCs in each of these fractions given the limited number of experimental animals, but it is possible to compare approximate numbers between the two cell populations. The haematopoietic (VE-cadherin-CD45+) fraction was able to reconstitute all five of the animals tested so we can predict that there were more than five HSCs present in the transplanted fraction. However, only four out of the five recipients of the DP cell population were reconstituted, indicating a frequency of fewer than five HSCs in that fraction. The observation that the level of reconstitution in each recipient transplanted with the haematopoietic fraction was, on average twofold higher than with the DP cells (Table 2) supports these comparative predictions. Presuming that the competitive pressure from host and carrier cells in both experimental groups was similar, we conclude that the total number of HSC/RUs in the transplanted haematopoietic population was higher than in the DP population.
|
|
Definitive HSCs preserve VE-cadherin expression while travelling via the embryonic circulation
We then determined if loss of VE-cadherin in the HSC pool occurs before or
after liver colonization. Following erythrocyte depletion,
VE-cadherin+ and VE-cadherin- fractions were flow sorted
from E12.5 circulation and transplanted into irradiated mice. Although eight
out of 12 mice that received VE-cadherin+ cells showed 10-90%
leukocyte chimerism (average of 54%), only one out of the 10 recipients of the
VE-cadherin- cells were successfully repopulated
(Table 2). Therefore, as in the
E11.5 AGM and E12.5 YS the majority if not all of circulatory HSCs continue
expression of VE-cadherin before colonising the liver.
Progressive phenotypic divergence of HSCs from the endothelial compartment
The emergence of extra-hepatic HSCs occurs in close ontogenic relation with
the endothelial compartment. We therefore analysed how the HSCs and
endothelial compartments phenotypically diverge during crucial developmental
stages, in the E12.5 blood and the E13.5 liver.
In the E11.5 AGM region, TIE2 expression was observed in both endothelial
and DP populations, but not in haematopoietic cells
(Fig. 2B). A similar but less
pronounced tendency was observed in the E12.5 circulation
(Fig. 6B). However, in the
E13.5 liver a significant proportion of haematopoietic cells (15%) expressed
TIE2 (Fig. 6C), which is
consistent with the appearance of HSCs in this fraction and expression of TIE2
in foetal liver HSCs (Hsu et al.,
2000).
The vast majority of cells in the endothelial populations of the E11.5 AGM
region and the E13.5 liver expressed Flk1
(Shalaby et al., 1997) at low
level (Fig. 2A;
Fig. 6C). The majority of DP
cells also demonstrated a FLK1low phenotype in these locations.
Expression of FLK1 in circulatory cells was negligible. No marked FLK1
expression was observed in the haematopoietic population in any of the organs
analyzed.
PECAM1 was expressed to a certain extent in all of the cell fractions
during development (Fig. 2B;
Fig. 6B,C). However, in
haematopoietic cells PECAM1 was expressed at significantly lower level than in
the endothelial and the DP fractions. The DP population in all tissues showed
bimodal (PECAM1low and PECAM1high) staining profile.
Significant expression of PECAM1 in E12.5 circulation was observed only in the
DP fraction (Fig. 6B).
Interestingly, HSCs at the pre-definitive stage in the yolk sac and in the
adult bone marrow are PECAM1 positive
(Baumann et al., 2004).
Ac-LDL uptake was observed at high level by the endothelial and the DP fractions in all tested locations (Fig. 2B; Fig. 6B,C). Lower level of uptake was found in the haematopoietic fractions with the exception of the E13.5 liver which did not take up any Ac-LDL (Fig. 6C).
The endothelial populations in all of the organs investigated showed a strong expression of KIT. Both in the E11.5 AGM region and in the E13.5 liver the DP populations were also significantly enriched for KIT+ cells in comparison with haematopoietic cells (Fig. 2B; Fig. 6C). A significant proportion of cells within the DP fraction of the E12.5 circulation also expressed KIT+ cells (Fig. 6B). In general, the level of KIT expression increased over time among all cell fractions.
SCA1 expression is absent in the early yolk sac and only appears on the
emergence of definitive haematopoiesis (de
Bruijn et al., 2002; Lu et
al., 1996
). The endothelial and the DP fractions expressed SCA1 at
higher levels than the haematopoietic fraction. By E12.5 both the endothelial
and DP populations within circulation comprise substantial subsets of
SCA1high cells (Fig.
6B). The level of SCA1 expression in circulating DP cells is
remarkably high. In the E13.5 foetal liver SCA1 is expressed at elevated
levels in all three populations (Fig.
6C).
Within the E12.5 circulation and the E13.5 foetal liver MAC1 continues to mark both the DP and the haematopoietic populations (Fig. 6B,C). MAC1 is fully excluded from the endothelial compartment of the AGM region, peripheral blood or liver (Fig. 2B; Fig. 6B,C).
In the E11.5 AGM region 4-integrin expression marks
3% of
endothelial cells but in the DP and haematopoietic populations virtually all
cells express
4-integrin. In the E12.5 circulation, the expression in
the endothelial fractions is absent and is restricted to
52% of the DP
population (Fig. 6B). In the
E13.5 liver the proportion of
4-integrin-expressing cells is markedly
increased in all three populations (Fig.
6C).
Very few cells in the E11.5 AGM were CD41 positive (Fig. 2B). In the E12.5 circulation CD41 expression in the DP and the haematopoietic fractions was markedly increased (Fig. 6B). A further increase in CD41 representation was observed in the endothelial, haematopoietic and DP populations of the E13.5 liver (Fig. 6C). Growth in numbers of CD41 haematopoietic cells during development may correlate with progressive production of megakaryocytes, or with the broad expression in embryonic haematopoietic cells.
Developmental downregulation of VE-cadherin within the HSC pool is not dependent on the liver microenvironment
To determine whether direct contact with the liver was required for the
loss of VE-cadherin expression, we explored whether VE-cadherin-
HSCs would develop in organ explants in vitro.
We therefore used an organ culture model which we have previously shown to
be permissive for expansion of HSC in the E11.5 AGM region and E12.5 YS
(Kumaravelu et al., 2002),
thus allowing us to test whether VE-cadherin-negative HSCs would develop
independently of the liver micro environment.
We found that by the end of the culture period, significant fractions of VE-cadherin-negative HSCs emerged in both the isolated AGM and the yolk sac (Table 4, see also Fig. S2 in the supplementary material). The proportions of mice reconstituted with VE-cadherin+ and VE-cadherin- AGM fractions were similar (seven out of 11 mice and seven out of nine mice reconstituted, respectively). Efficient repopulation was also observed with yolk sac VE-cadherin+ and VE-cadherin- fractions (seven out of 10 mice and 10 out of 10 mice, respectively) (Table 4). Assuming that the ex vivo generation of HSCs reflects the in vivo pathway with reasonable fidelity, it is therefore likely that the downregulation of VE-cadherin within the HSC pool is a function of an intrinsic developmental time. This process, as hypothesised by the authors, is presented in Movie 1 (see supplementary material).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The idea that the haematopoietic and endothelial systems have a common
cellular origin was originally based on: (1) morphological similarities
observed between developing haematopoietic and endothelial cells in YS blood
islands (Sabin, 1920), (2)
clusters of haematopoietic cells that are believed to be budding from the
floor of the dorsal aorta; and (3) a large number of shared genetic markers
(Godin and Cumano, 2002
).
Haematopoietic development via an endothelial differentiation pathway has been
analyzed experimentally using in vitro differentiation assays based on Flk1
and VE-cadherin expression (Chung et al.,
2002
; Kennedy et al.,
1997
; Lacaud et al.,
2002
; Nishikawa et al.,
1998a
; Nishikawa et al.,
1998b
). Cells with haemangioblastic characteristics have been
reported in the early primitive streak
(Huber et al., 2004
) and the
adult haematopoietic system (Bailey et al.,
2004
; Pelosi et al.,
2002
).
We have focused on cells co-expressing VE-cadherin and CD45, reasoning that
if the definitive haematopoietic system originates from the embryonic
endothelium or the haemangioblast, then the first founder HSC may inherit
endothelial features. Previous data corroborate this idea
(de Bruijn et al., 2002;
North et al., 2002
). We have
followed here the evolution of the dual endothelial and haematopoietic
character of highly repopulating definitive HSCs through key developmental
stages: (1) initiation in the E11.5 AGM and E12.5 YS; (2) circulation in
peripheral blood (E12.5); and (3) settling in the foetal liver (E13.5).
VE-cadherin and CD45 are predominantly mutually exclusive with DP cells representing a minority of about 0.05% in embryonic haematopoietic tissues. We used flow cytometric cell sorting to purify populations based on VE-cadherin and CD45 expression and, using in vitro methylcellulose assay found that the highest frequency of haematopoietic progenitors was present in the DP population. We also confirmed that long-term highly repopulating HSCs in the E11.5 AGM reside within this fraction. Furthermore, at the peak of stem cell activity in the E12.5 YS, HSCs also reside within the VE-cadherin expressing fraction. The enrichment of HSCs in the DP populations is very high: the E11.5 AGM and E12.5 YS contain one HSC in 70 and one HSC in 64 DP cells, respectively. Thus, HSC emergence in both extra-hepatic haematopoietic organs is associated with VE-cadherin expression.
Further phenotypic characterization revealed striking similarity between
the DP and endothelial cells in the E11.5 AGM region. Both the endothelial and
DP populations are largely
TIE2+FLK1+PECAM1+Ac-LDL-receptor+.
The haematopoietic fractions are either negative for these markers or express
them at low level. The analysis of other `HSC markers' has shown that the DP
fraction contains particularly high levels of KIT, SCA1, CD34 and AA4.1.
Endothelial cells also but to a lesser extent are enriched for these markers.
Close similarity between the endothelium and the DP fraction of the E11.5 AGM
region, with respect to the above markers, again strongly suggests an
ontogenetic link between these populations. However, in contrast to true
AGM-derived endothelial cells, the DP cells did not form endothelial tubules
in vitro, suggesting their functional divergence from the vasculature.
Furthermore, by E11.5 endothelial cells are no longer capable of producing
haematopoietic cells in vitro. By this stage, haematopoietic differentiation
is associated exclusively with the DP fraction, whereas during E8-E10, such
activity is clearly associated with the endothelial fraction
(Fraser et al., 2003). Thus,
despite phenotypic similarity between the endothelial and DP populations, a
clear functional divergence develops in the embryo. The divergence of the
endothelial and the DP cells can also be observed at the morphological level,
with the DP fraction being enriched for cells with a blast-like
morphology.
It is interesting that the DP population is entirely positive for an early
embryonic HSC marker MAC1+
(Morrison et al., 1995;
Sanchez et al., 1996
), which
is entirely absent from the endothelial fraction, suggesting that MAC1 marks a
diverging point between the segregating endothelial and HSC/haematopoietic
compartments.
We then tested if HSCs that moved away from their site of origin lose their endothelial character. We have found that long-term repopulating HSCs which enter the circulation by E12.5 remain VE-cadherin+. The DP cells continue expressing TIE2, PECAM1 and Ac-LDL receptor, but expression of FLK1 becomes negligible. Among stem cell markers, SCA1 is expressed at particularly high level and the expression of a foetal HSC marker MAC1 is also remarkably high (Fig. 6C).
By E13.5 HSC activity within the foetal liver becomes associated
predominantly, but not entirely, with the VE-cadherin negative fraction. [The
discrepancy with the previous report
(North et al., 2002) may
result from differences in gating strategies or sensitivities of the flow
cytometers used.] The phenotypic similarities between the endothelial and the
DP fractions remain evident but expression of Ac-LDL receptor becomes
significantly lower in the DP cells (Fig.
7B). Interestingly, a fraction of cells in the haematopoietic
population, which contains most liver HSCs, becomes TIE2+,
consistent with previous reports on TIE2 expression in HSCs
(Arai et al., 2004
;
Hsu et al., 2000
). In
accordance with this, stem cell markers (SCA1 and KIT) are also upregulated in
the haematopoietic fraction in the E13.5 liver (compared with the
haematopoietic fractions in the AGM region and circulation).
As downregulation of VE-cadherin in the liver occurs only at the
post-migratory stage, we tested if direct contact with the liver was required
for VE-cadherin downregulation. To this end we analysed the in vitro
development of HSCs in isolated E11.5 AGM region and E12.5 YS using an organ
culture system (Kumaravelu et al.,
2002). By the end of the culture period at least 50% of HSCs lost
VE-cadherin expression. Therefore, progressive divergence of HSCs from the
endothelial compartment does not require specific contact with the foetal
liver and probably depends on developmental time. In line with this, the
analysis of the advanced E12.5 AGM region in vivo has also shown that
VE-cadherin is progressively downregulated in HSCs with time.
We also tested multi-lineage contribution of VE-cadherin+ and VE-cadherin- foetal liver HSCs into recipient haematopoietic system. Although no obvious differences have been observed between these two cell types, it needs to be elucidated further if the emergence of `advanced' VE-cadherin-CD45+ HSCs is associated with the appearance of some novel functional characteristics.
In summary, we show that definitive HSCs emerging in pre-liver embryonic sites are uniquely promiscuous. Not only does this fraction co-express the predominantly mutually exclusive VE-cadherin and CD45 determinants, but it also bears cardinal markers of endothelial and haematopoietic/stem cell differentiation. We show that this promiscuity is largely retained in circulatory HSCs. However, after seeding the foetal liver, a significant proportion of HSCs lose their endothelial identity. We show that this process does not depend on immediate contact with the liver, but rather appears to be a function of developmental time.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/18/4179/DC1
* These authors contributed equally to this work
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arai, F., Hirao, A., Ohmura, M., Sato, H., Matsuoka, S., Takubo, K., Ito, K., Koh, G. Y. and Suda, T. (2004). Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 118,149 -161.[CrossRef][Medline]
Arroyo, A. G., Yang, J. T., Rayburn, H. and Hynes, R. O. (1996). Differential requirements for alpha4 integrins during fetal and adult hematopoiesis. Cell 85,997 -1008.[CrossRef][Medline]
Bailey, A. S. and Fleming, W. H. (2003). Converging roads: evidence for an adult hemangioblast. Exp. Hematol. 31,987 -993.[CrossRef][Medline]
Bailey, A. S., Jiang, S., Afentoulis, M., Baumann, C. I.,
Schroeder, D. A., Olson, S. B., Wong, M. H. and Fleming, W. H.
(2004). Transplanted adult hematopoietic stems cells
differentiate into functional endothelial cells. Blood
103, 13-19.
Baron, M. H. (2003). Embryonic origins of mammalian hematopoiesis. Exp. Hematol. 31,1160 -1169.[CrossRef][Medline]
Baumann, C. I., Bailey, A. S., Li, W., Ferkowicz, M. J., Yoder,
M. C. and Fleming, W. H. (2004). PECAM-1 is expressed on
hematopoietic stem cells throughout ontogeny and identifies a population of
erythroid progenitors. Blood
104,1010
-1016.
Bertrand, J. Y., Giroux, S., Golub, R., Klaine, M., Jalil, A.,
Boucontet, L., Godin, I. and Cumano, A. (2005).
Characterization of purified intraembryonic hematopoietic stem cells as a tool
to define their site of origin. Proc. Natl. Acad. Sci.
USA 102,134
-139.
Breier, G., Breviario, F., Caveda, L., Berthier, R., Schnurch,
H., Gotsch, U., Vestweber, D., Risau, W. and Dejana, E.
(1996). Molecular cloning and expression of murine vascular
endothelial-cadherin in early stage development of cardiovascular system.
Blood 87,630
-641.
Carmeliet, P., Lampugnani, M. G., Moons, L., Breviario, F., Compernolle, V., Bono, F., Balconi, G., Spagnuolo, R., Oostuyse, B., Dewerchin, M. et al. (1999). Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis. Cell 98,147 -157.[CrossRef][Medline]
Chen, C. Z., Li, L., Li, M. and Lodish, H. F. (2003). The endoglin (positive) sca-1 (positive) rhodamine (low) phenotype defines a near-homogeneous population of long-term repopulating hematopoietic stem cells. Immunity 19,525 -533.[CrossRef][Medline]
Christensen, J. L., Wright, D. E., Wagers, A. J. and Weissman, I. L. (2004). Circulation and chemotaxis of fetal hematopoietic stem cells. PLoS Biol. 2, E75.[CrossRef][Medline]
Chung, Y. S., Zhang, W. J., Arentson, E., Kingsley, P. D.,
Palis, J. and Choi, K. (2002). Lineage analysis of the
hemangioblast as defined by FLK1 and SCL expression.
Development 129,5511
-5520.
de Bruijn, M. F., Speck, N. A., Peeters, M. C. and Dzierzak,
E. (2000). Definitive hematopoietic stem cells first develop
within the major arterial regions of the mouse embryo. EMBO
J. 19,2465
-2474.
de Bruijn, M. F. T. R., Ma, X., Robin, C., Ottersbach, K., Sanchez, M.-J. and Dzierzak, E. (2002). Hematopoietic stem cells localize to the endothelial cell layer in the midgestation mouse aorta. Immunity 16,673 -683.[CrossRef][Medline]
Dzierzak, E. and Medvinsky, A. (1995). Mouse embryonic hematopoiesis. Trends Genet. 11,359 -366.[CrossRef][Medline]
Ema, H. and Nakauchi, H. (2000). Expansion of
hematopoietic stem cells in the developing liver of a mouse embryo.
Blood 95,2284
-2288.
Ema, M. and Rossant, J. (2003). Cell fate decisions in early blood vessel formation. Trends Cardiovasc. Med. 13,254 -259.[CrossRef][Medline]
Emambokus, N. R. and Frampton, J. (2003). The glycoprotein IIb molecule is expressed on early murine hematopoietic progenitors and regulates their numbers in sites of hematopoiesis. Immunity 19,33 -45.[CrossRef][Medline]
Fehling, H. J., Lacaud, G., Kubo, A., Kennedy, M., Robertson,
S., Keller, G. and Kouskoff, V. (2003). Tracking mesoderm
induction and its specification to the hemangioblast during embryonic stem
cell differentiation. Development
130,4217
-4227.
Ferkowicz, M. J., Starr, M., Xie, X., Li, W., Johnson, S. A.,
Shelley, W. C., Morrison, P. R. and Yoder, M. C. (2003). CD41
expression defines the onset of primitive and definitive hematopoiesis in the
murine embryo. Development
130,4393
-4403.
Fraser, S. T., Ogawa, M., Yu, R. T., Nishikawa, S., Yoder, M. C. and Nishikawa, S.-I. (2002). Definitive hematopoietic commitment within the embryonic vascular endothelial-cadherin (+) population. Exp. Hematol. 30,1070 -1078.[CrossRef][Medline]
Fraser, S. T., Ogawa, M., Yokomizo, T., Ito, Y. and Nishikawa, S. (2003). Putative intermediate precursor between hematogenic endothelial cells and blood cells in the developing embryo. Dev. Growth Differ. 45,63 -75.[CrossRef][Medline]
Fujimoto, T., Ogawa, M., Minegishi, N., Yoshida, H., Yokomizo,
T., Yamamoto, M. and Nishikawa, S. (2001). Step-wise
divergence of primitive and definitive haematopoietic and endothelial cell
lineages during embryonic stem cell differentiation. Genes
Cells 6,1113
-1127.
Gekas, C., Dieterlen-Lievre, F., Orkin, S. H. and Mikkola, H. K. (2005). The Placenta Is a Niche for Hematopoietic Stem Cells. Dev. Cell 8,365 -375.[CrossRef][Medline]
Gerber, H. P., Malik, A. K., Solar, G. P., Sherman, D., Liang, X. H., Meng, G., Hong, K., Marsters, J. C. and Ferrara, N. (2002). VEGF regulates haematopoietic stem cell survival by an internal autocrine loop mechanism. Nature 417,954 -958.[CrossRef][Medline]
Godin, I. and Cumano, A. (2002). The hare and the tortoise: an embryonic haematopoietic race. Nat. Rev. Immunol. 2,593 -604.[Medline]
Hsu, H. C., Ema, H., Osawa, M., Nakamura, Y., Suda, T. and
Nakauchi, H. (2000). Hematopoietic stem cells express Tie-2
receptor in the murine fetal liver. Blood
96,3757
-3762.
Huber, T. L., Kouskoff, V., Fehling, H. J., Palis, J. and Keller, G. (2004). Haemangioblast commitment is initiated in the primitive streak of the mouse embryo. Nature 432,625 -630.[CrossRef][Medline]
Ikuta, K. and Weissman, I. L. (1992). Evidence
that hematopoietic stem cells express mouse c-kit but do not depend on steel
factor for their generation. Proc. Natl. Acad. Sci.
USA 89,1502
-1506.
Jaffredo, T., Gautier, R., Eichmann, A. and Dieterlen-Lievre,
F. (1998). Intraaortic hemopoietic cells are derived from
endothelial cells during ontogeny. Development
125,4575
-4583.
Jaffredo, T., Gautier, R., Brajeul, V. and Dieterlen-Lievre, F. (2000). Tracing the progeny of the aortic hemangioblast in the avian embryo. Dev. Biol. 224,204 -214.[CrossRef][Medline]
Jordan, H. E. (1917). Aortic cell clusters in
vertebrate embryos. Proc. Natl. Acad. Sci. USA
3, 149-156.
Kennedy, M., Firpo, M., Choi, K., Wall, C., Robertson, S., Kabrun, N. and Keller, G. (1997). A common precursor for primitive erythropoiesis and definitive haematopoiesis. Nature 386,488 -493.[CrossRef][Medline]
Kondo, M., Wagers, A. J., Manz, M. G., Prohaska, S. S., Scherer, D. C., Beilhack, G. F., Shizuru, J. A. and Weissman, I. L. (2003). Biology of hematopoietic stem cells and progenitors: implications for clinical application. Annu. Rev. Immunol. 21,759 -806.[CrossRef][Medline]
Kumaravelu, P., Hook, L., Morrison, A. M., Ure, J., Zhao, S.,
Zuyev, S., Ansell, J. and Medvinsky, A. (2002). Quantitative
developmental anatomy of definitive haematopoietic stem cells/long-term
repopulating units (HSC/RUs): role of the aorta-gonad-mesonephros (AGM) region
and the yolk sac in colonisation of the mouse embryonic liver.
Development 129,4891
-4899.
Lacaud, G., Gore, L., Kennedy, M., Kouskoff, V., Kingsley, P.,
Hogan, C., Carlsson, L., Speck, N., Palis, J. and Keller, G.
(2002). Runx1 is essential for hematopoietic commitment at the
hemangioblast stage of development in vitro. Blood
100,458
-466.
Lu, L. S., Wang, S. J. and Auerbach, R. (1996).
In vitro and in vivo differentiation into B cells, T cells, and myeloid cells
of primitive yolk sac hematopoietic precursor cells expanded >100-fold by
coculture with a clonal yolk sac endothelial cell line. Proc. Natl.
Acad. Sci. USA 93,14782
-14787.
Medvinsky, A. and Dzierzak, E. (1996). Definitive hematopoiesis is autonomously initiated by the AGM region. Cell 86,897 -906.[CrossRef][Medline]
Medvinsky, A. L., Gan, O. I., Semenova, M. L. and Samoylina, N.
L. (1996). Development of day-8 colony-forming unit-spleen
hematopoietic progenitors during early murine embryogenesis: spatial and
temporal mapping. Blood
87,557
-566.
Mikkola, H. K., Fujiwara, Y., Schlaeger, T. M., Traver, D. and
Orkin, S. H. (2003). Expression of CD41 marks the initiation
of definitive hematopoiesis in the mouse embryo. Blood
101,508
-516.
Mitjavila-Garcia, M. T., Cailleret, M., Godin, I., Nogueira, M. M., Cohen-Solal, K., Schiavon, V., Lecluse, Y., Le Pesteur, F., Lagrue, A. H. and Vainchenker, W. (2002). Expression of CD41 on hematopoietic progenitors derived from embryonic hematopoietic cells. Development 129,2003 -2013.[Medline]
Moore, M. A. and Metcalf, D. (1970). Ontogeny of the haemopoietic system: yolk sac origin of in vivo and in vitro colony forming cells in the developing mouse embryo. Br. J. Haematol. 18,279 -296.[Medline]
Morrison, S. J., Hemmati, H. D., Wandycz, A. M. and Weissman, I.
L. (1995). The purification and characterization of fetal
liver hematopoietic stem cells. Proc. Natl. Acad. Sci.
USA 92,10302
-10306.
Muller, A. M., Medvinsky, A., Strouboulis, J., Grosveld, F. and Dzierzak, E. (1994). Development of hematopoietic stem cell activity in the mouse embryo. Immunity 1, 291-301.[CrossRef][Medline]
Nishikawa, S. I., Nishikawa, S., Hirashima, M., Matsuyoshi, N.
and Kodama, H. (1998a). Progressive lineage analysis by cell
sorting and culture identifies FLK1+VE-cadherin+ cells at a diverging point of
endothelial and hemopoietic lineages. Development
125,1747
-1757.
Nishikawa, S. I., Nishikawa, S., Kawamoto, H., Yoshida, H., Kizumoto, M., Kataoka, H. and Katsura, Y. (1998b). In vitro generation of lymphohematopoietic cells from endothelial cells purified from murine embryos. Immunity 8, 761-769.[CrossRef][Medline]
North, T., Gu, T. L., Stacy, T., Wang, Q., Howard, L., Binder,
M., Marin-Padilla, M. and Speck, N. A. (1999). Cbfa2 is
required for the formation of intra-aortic hematopoietic clusters.
Development 126,2563
-2575.
North, T. E., de Bruijn, M. F., Stacy, T., Talebian, L., Lind, E., Robin, C., Binder, M., Dzierzak, E. and Speck, N. A. (2002). Runx1 expression marks long-term repopulating hematopoietic stem cells in the midgestation mouse embryo. Immunity 16,661 -672.[CrossRef][Medline]
Ogawa, M., Kizumoto, M., Nishikawa, S., Fujimoto, T., Kodama, H.
and Nishikawa, S. I. (1999). Expression of alpha4-integrin
defines the earliest precursor of hematopoietic cell lineage diverged from
endothelial cells. Blood
93,1168
-1177.
Ottersbach, K. and Dzierzak, E. (2005). The murine placenta contains hematopoietic stem cells within the vascular labyrinth region. Dev. Cell 8, 377-387.[CrossRef][Medline]
Pelosi, E., Valtieri, M., Coppola, S., Botta, R., Gabbianelli,
M., Lulli, V., Marziali, G., Masella, B., Muller, R., Sgadari, C. et al.
(2002). Identification of the hemangioblast in postnatal life.
Blood 100,3203
-3208.
Petrenko, O., Beavis, A., Klaine, M., Kittappa, R., Godin, I. and Lemischka, I. R. (1999). The molecular characterization of the fetal stem cell marker AA4. Immunity 10,691 -700.[CrossRef][Medline]
Sabin, F. R. (1920). Studies on the origin of blood vessels and of red blood corpuscules as seen in the living blastoderm of chicks during the second day of incubation. Contrib. Embryol. 9,213 -262.
Sanchez, M. J., Holmes, A., Miles, C. and Dzierzak, E. (1996). Characterization of the first definitive hematopoietic stem cells in the AGM and liver of the mouse embryo. Immunity 5,513 -525.[CrossRef][Medline]
Shalaby, F., Ho, J., Stanford, W. L., Fischer, K. D., Schuh, A. C., Schwartz, L., Bernstein, A. and Rossant, J. (1997). A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis. Cell 89,981 -990.[CrossRef][Medline]
Takakura, N., Huang, X. L., Naruse, T., Hamaguchi, I., Dumont, D. J., Yancopoulos, G. D. and Suda, T. (1998). Critical role of the TIE2 endothelial cell receptor in the development of definitive hematopoiesis. Immunity 9, 677-686.[CrossRef][Medline]
Tavian, M., Coulombel, L., Luton, D., Clemente, H. S.,
Dieterlen-Lievre, F. and Peault, B. (1996). Aorta-associated
CD34+ hematopoietic cells in the early human embryo.
Blood 87,67
-72.
Tavian, M., Hallais, M. F. and Peault, B.
(1999). Emergence of intraembryonic hematopoietic precursors in
the pre-liver human embryo. Development
126,793
-803.
Toles, J. F., Chui, D. H., Belbeck, L. W., Starr, E. and Barker,
J. E. (1989). Hemopoietic stem cells in murine embryonic yolk
sac and peripheral blood. Proc. Natl. Acad. Sci. USA
86,7456
-7459.
Uchida, N. and Weissman, I. L. (1992).
Searching for hematopoietic stem cells: evidence that Thy-1.1lo Lin-Sca-1+
cells are the only stem cells in C57BL/Ka-Thy-1.1 bone marrow. J.
Exp. Med. 175,175
-184.
Weissman, I., Pappaioannou, V. and Gardner, R. (1978). Fetal haematopoietic origins of the adult hematolymphoid system. In Cold Spring Harbor Meeting on Differentiation of Normal and Neoplastic Hematopoietic Cells (ed. B. Clarkson, P. A. Marks and J. E. Till), pp. 33-47. Cold Spring Harbor. Cold Spring Harbor Laboratory Press.
Yamaguchi, T. P., Dumont, D. J., Conlon, R. A., Breitman, M. L.
and Rossant, J. (1993). flk-1, an flt-related receptor
tyrosine kinase is an early marker for endothelial cell precursors.
Development 118,489
-498.
Yoder, M. C., Hiatt, K., Dutt, P., Mukherjee, P., Bodine, D. M. and Orlic, D. (1997). Characterization of definitive lymphohematopoietic stem cells in the day 9 murine yolk sac. Immunity 7,335 -344.[CrossRef][Medline]
Yuasa, H., Takakura, N., Shimomura, T., Suenobu, S., Yamada, T., Nagayama, H., Oike, Y. and Suda, T. (2002). Analysis of human TIE2 function on hematopoietic stem cells in umbilical cord blood. Biochem. Biophys. Res. Commun. 298,731 -737.[CrossRef][Medline]