1 Laboratoire d'Embryologie cellulaire et moléculaire du CNRS et du
Collège de France, 49 bis, avenue de la Belle Gabrielle, 94736 Nogent
s/Marne cedex, France
2 Laboratório de Neurobiologia e Hematologia Celular e Molecular, BEG,
CCB, Universidade Federal de Santa Catarina, Campus Universitário,
88040-900, Florianópolis, SC, Brazil
* Author for correspondence (e-mail: fdieterlen{at}college-de-france.fr)
Accepted 25 July 2003
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SUMMARY |
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Key words: Placenta, Hematopoiesis, Clonogenic progenitors, Mouse
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Introduction |
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In the avian embryo, a second appendage (the allantois) was shown to
produce hematopoietic progenitors (Caprioli
et al., 1998; Caprioli et al.,
2001
). Because the mouse chorioallantoic placenta develops by
fusion of the allantois to the ectoplacental cone, we asked whether the
placenta might participate in mouse fetal hematopoiesis and probed it for the
presence of hematopoietic progenitors. The mouse placenta has previously been
shown to harbor B-cell progenitors
(Melchers, 1979
). We initiated
our investigation around the stage of fusion of the allantois to the
ectoplacental cone, which occurs in embryos with seven somite pairs (sp) or
thereabouts [at embryonic days 8-9 (E8-E9)]
(Theiler, 1972
;
Downs, 2002
).
Here, we report a developmental analysis comparing the types and frequency
of in vitro clonogenic progenitors
(Metcalf et al., 1979)
detected in the placenta, yolk sac, early embryo and fetal liver, every two
days from E8 to E17. We could show that: (1) the placenta was 2-4 times richer
in myeloid progenitors than the fetal liver; (2) these progenitors were
derived from the fetal, not the maternal, compartment; (3) by E9 and E10, the
placenta already harbored progenitors, whereas the liver did not; (4) at each
stage studied, high-proliferation-potential colony-forming cells (HPP-CFCs),
which were able to give rise to subcolonies of the same type up to 60 days,
were several times more frequent in placenta than in fetal liver or yolk sac.
HPP-CFCs are defined as multilineage colonies grown in semisolid medium whose
diameter exceeds 0.5 mm (Pohlmann et al.,
2001
).
To confirm the initiation of hematopoiesis, we analysed the expression of
several genes that are involved in the emergence of hematopoietic activity, at
the 18 and 20 sp stages. The Runx1 gene encodes the DNA binding
subunit of a transcription factor belonging to the core binding factor (CBF)
family (Wang et al., 1996).
Its expression is rigorously restricted to the sites where HSCs become
committed in the embryo (North et al.,
1999
; North et al.,
2002
; Bollerot and Jaffredo, 2003), thus representing a distinct
sign of HSC emergence. The Scl/Tal1 (Tal1
Mouse Genome Informatics) gene is required for the development of all
hematopoietic lineages (Porcher et al.,
1996
; Robb et al.,
1996
). Finally the Tel/Etv6 gene activity is
required for the appearance of hematopoietic stem cell activity in the bone
marrow (Wang et al.,
1998
).
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Materials and methods |
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CFC and HPP-CFC assays
For analysis of progenitor activity in each organ, 104 cells
pooled from between four and six embryos were plated per well in LabTek
chambers (four wells) in 500 µl MethoCult M3434 methylcellulose media (Stem
Cell Technologies, Vancouver, BC, Canada). The commercial medium contains
methylcellulose in IMDM [15% fetal calf serum (FCS), 1% bovine serum albumin
(BSA), 104 M 2-mercaptoethanol, 2 mM glutamine, 3 U
ml1 recombinant human (rh) erythropoietin, 10 ng
ml1 rh interleukin 6 (IL-6), 10 ng ml1
recombinant mouse (rm) IL-3, 50 ng ml1 rm Stem Cell Factor
(SCF), 10 µg ml1 rh insulin and 200 µg
ml1 human transferrin (iron saturated)]. After 14 days of
culture at 37°C in a humid atmosphere containing 5% CO2,
colonies were identified based on their morphology under an inverted
microscope and scored. Each value was obtained from the means of three or four
independent assays. Individual colonies were plucked using fine-drawn Pasteur
pipettes and May-Grünwald-Giemsa stained. In some experiments, the cells
were obtained from GFP+ embryos in which only the fetal
contribution to the placenta was transgenic.
To check for the presence of long-term HPP-CFCs, first passage colonies with a diameter exceeding 0.5 mm were plucked from eight wells after 20-25 days of culture, pooled, washed with PBS. Cells were counted and 104 cells were replated per well in MethoCult M3434 medium. The colonies were replated three times according to the same protocol and scored 60 days after initial seeding.
Statistical analysis
Mann-Whitney U non-parametric analysis was performed using Instat
(GraphPad Software, San Diego, CA).
RNA extraction and RT-PCR
Total RNA was extracted from cells obtained from tissues with GenElute
Mammalian Total RNA Kit (Sigma). For reverse-transcription polymerase chain
reaction (RT-PCR), first-strand cDNA was synthesized with the use of anchored
oligo(dT)23 and enhanced AMV reverse transcriptase (Sigma) from 0.5
µg total RNA. Specific cDNA was amplified with Taq DNA polymerase (PCR
Master Mix, Promega) using pairs of oligonucleotide primers as follows:
Samples were denatured at 94°C for 5 minutes, followed by 35 amplification rounds, each consisting of 94°C for 30 seconds (denaturing), 60°C for 1 minute (annealing) and 72°C for 3 minutes (extension). Products were separated on a 1.2% agarose gel, stained with ethidium bromide and photographed.
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Results |
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Furthermore, many placental CFU-GEMMs were huge and so qualified more adequately as HPP-CFCs, with a very large core of hemoglobinized cells surrounded by dense clusters of granulocytes (Fig. 4A,D). Yolk sac and liver colonies were never that large (Fig. 4B,C). On cytological examination, both yolk sac and placental colonies were very rich in erythroblasts (Fig. 4D,E), yolk sac was significantly enriched in late erythroblasts (Fig. 4E), liver displayed erythroid cells expelling their nucleus (Fig. 4F), and megakaryocytes were especially abundant in placental CFU-GEMMs (not shown).
|
The frequency of CFU-GMs varied differently from that of other colony
types. Placental progenitors peaked at E12, liver frequency increased steadily
during the period studied and yolk sac frequency remained stable
(Fig. 3). E10 liver, again, did
not contain any CFU-GMs, with the exception of one colony in four independent
assays (i.e. 16 wells). Placenta and yolk sac CFU-GMs were dense, medium to
large sized, and often actually so large that they seemed to result from the
fusion of clusters (data not shown). The time course of colony emergence in
the embryo caudal half fitted with previous studies about the
para-aortic-splanchnopleura (P-Sp) (Godin
et al., 1995).
All in all, the salient points in this developmental pattern are that: (1) the appearance of clonogenic progenitors in the placenta lagged slightly behind that in the embryo proper; (2) progenitors were 2-4 times more frequent in the placenta than in the liver; (3) placental colonies had, at all times, a more immature phenotype than these from yolk sac or liver. The differences in frequency and immaturity were especially striking at E15 and E17.
Cellularity of the organs and total number of progenitors in each
organ
The total number of nucleated cells nearly doubled in the placenta and yolk
sac from E10 to E17. By contrast, the number of cells in the liver increased
about 13-fold between E12 and E17 (Table
2). From these counts, it is possible to evaluate the total number
of progenitors in each organ at these developmental stages
(Table 3).
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Long-term HPP-CFCs
In order to monitor the self-renewal capacity of hematopoietic progenitors
in the various organs, we replated the very large multilineage colonies
derived from HPP-CFCs every 2 weeks for 2-3 months. These experiments were
carried out using E12, E15 or E17 unsorted cell preparations. At each
replating, some very large colonies were again obtained. When the colonies
were scored at 60 days, 4.5 CFU-GEMMs were obtained from 104
replated placental cells. Similar results were observed whether cells were
obtained from E12, E15 or E17 embryos (Fig.
5, E15). By contrast, HPP-CFCs were rarely obtained from the yolk
sac. Liver progenitors did give rise to this type of colony, but the frequency
was half that in the placenta. Furthermore, the liver colonies were smaller
than the placental colonies. Both parameters thus indicate a progressive
extinction of the self-renewal capacity in the liver.
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Discussion |
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It was important to verify that the progenitors were derived from the fetal
part of the placenta. Melchers used different major histocompatibility
haplotypes to distinguish maternal from fetal cells and concluded that the B
lymphoid progenitors detected were indeed of fetal origin
(Melchers, 1979). Using a
green fluorescent protein gene as a marker, we extend this finding to myeloid
progenitors.
The results we report here do not give clues about whether the progenitors
found in the placenta originate in situ or colonize the appendage. Although
the yolk sac produces its own stem cells, the fetal liver and bone marrow are
known to become seeded by extrinsic stem cells, a sine qua non condition for
their hematopoietic activity to switch on
(Metcalf and Moore, 1971;
Jotereau and Le Douarin, 1978
;
Johnson and Moore, 1975
;
Houssaint, 1981
;
Pardanaud et al., 1989
).
Although progenitors emerging in the yolk sac might contribute to the seeding
of definitive hemopoietic organs (in a more or less transitory fashion,
depending on species) (Weissman et al.,
1978
; Beaupain et al.,
1979
; Kau and Turpen,
1983
; Pardanaud et al.,
1989
; Yoder et al.,
1997
; Palis et al.,
1999
), the secondary wave of progenitors, which becomes committed
in the embryo, has been recognized as a source of definitive hematopoiesis, in
bird (Dieterlen-Lièvre,
1975
), amphibian (Turpen et
al., 1981
; Ciau-Uitz et al.,
2000
), mouse (Godin et al.,
1993
; Godin et al.,
1995
; Cumano et al.,
1996
; Medvinsky and Dzierzak,
1996
) and human (Tavian et
al., 1996
) embryos.
The P-SP/AGM (Godin et al.,
1993; Medvinsky et al.,
1993
; Cumano et al.,
1996
; Medvinsky and Dzierzak,
1996
) is accepted as the region where these intraembryonic HSCs
become committed (Garcia-Porrero et al.,
1995
; Garcia-Porrero et al.,
1998
; Wood et al.,
1997
; Jaffredo et al.,
1998
; Jaffredo et al.,
2000
; de Bruijn et al.,
2002
; North et al.,
2002
; Oberlin et al.,
2002
). The AGM is at present considered to be responsible for the
production of HSCs colonizing the fetal liver. In this region, the hemogenic
activity begins around the 15 sp stage in the P-Sp
(Godin et al., 1995
) and
continues in the AGM, where it peaks around the stage of 40 sp and thereafter
declines (Medvinsky et al.,
1993
). It is intriguing that the emergence of progenitors in the
placenta follows shortly after the emergence of progenitors in the embryonic
aorta, vitelline and umbilical arteries. The progenitor producing activity of
the placenta might represent a spatial extension of the activity ongoing in
these arteries, which has previously been described by several authors
(de Bruijn et al., 2000
;
North et al., 2002
;
Tavian et al., 1999
).
The appearance of placental progenitors and the first expression of the
transcription factors Scl/tal1 and Runx1 are simultaneous. The products of
these genes are required for definitive HSC commitment
(Porcher et al., 1996;
North et al., 1999
;
North et al., 2002
),
suggesting that this event might occur in the placenta. The third gene whose
expression was analyzed, Tel/Etv6, was selected for study
because of the mutant phenotype: no
Tel/Etv6/ progenitors colonize the
bone marrow of chimeras, whereas these progenitors thrive in the yolk sac and
fetal liver (Wang et al.,
1998
). A more detailed expression pattern over a longer period of
development than was analyzed in the present work will be needed to understand
how placental hematopoiesis is initiated.
A possible erythropoietic function of the mouse allantois, which is known
to contribute blood vessels to the placenta
(de Bruijn et al., 2002), was
previously explored without success (Downs
and Harmann, 1997
; Downs et
al., 1998
). Vasculogenesis, a process often associated with the
production of blood progenitors, occurred in the early rudiment cultured in
vitro (Downs et al., 1998
;
Drake and Fleming, 2000
) but
no definite signs of erythropoiesis could be ascertained
(Downs et al., 1998
). However,
in these experiments, the allantoises were explanted from head-fold-stage
embryos, cultured until the 13-15 sp stages and scanned for benzidine-stained
erythrocytes. In the present investigation, clonogenic progenitors [detected
by their colony-forming potential in the presence of appropriate cytokines
(i.e. left for a further 14 days to differentiate)] were first found among
cells dissected from 18-20 sp stage embryos.
Because of the highly migratory nature of HSCs and progenitors, the riddle
of their origin is difficult to solve once the circulation is established.
Interestingly, the frequencies and types of progenitors found in the present
investigation are very different from one organ to the other. Such differences
have previously been described between human yolk sac and embryo
(Huyhn et al., 1995), and
argue against a substantial non-directional traffic of progenitors between
hematopoietic sites. Interestingly, in this respect, the E15-17 yolk sac still
harbors some CFU-GEMMs, suggesting that its hemogenic activity might be
sustained longer than previously thought. Among the three organs under
scrutiny here, the fetal liver differs from the placenta by having lower
frequencies of CFUGEMMs and HPP-CFCs, a much greater abundance of late
progenitors such as CFU-GMs and CFU-Es, and a higher cellularity. Thus, the
role of placental hematopoiesis is clearly different from that of the liver,
where hematopoietic cells multiply and differentiate.
In conclusion, the presence of abundant early clonogenic progenitors point
to the placenta as a major hematopoietic organ of the fetus. Human cord blood
progenitors at birth (Mayani and Lansdorp,
1998) are strikingly similar in frequency and potential to the
progenitors detected here in the mouse placenta. Together with the anatomical
and developmental relationships between umbilical cord and placenta, these
similarities make it likely that cord blood progenitors come from the
placenta.
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ACKNOWLEDGMENTS |
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