J. Embryol. exp. Morph. 73, 87-95, 1983
Printed in Great Britain © The Company of Biologists Limited 1983
g7
In vivo induction of granulopoiesis in visceral yolksac cells by foetal hepatic factors
By M. A. ANCKAERT 1 AND M. SYMANN 2
From the Unite de Recherches sur les Maladies du Sang, Laboratoire
d'Hematologie Experimentale, Universite Catholique de Louvain and Ludwig
Institute for Cancer Research (Brussels Branch)
SUMMARY
In order to evaluate the hypothetical activity of foetal hepatic factors on putative yolk-sac
haemopoietic stem cells we used the Double Diffusion Chamber (DDC) technique. The DDC
were made of a regulator compartment, where foetal hepatic tissue was introduced and a test
compartment where visceral yolk-sac cells were cultured. In this system a hepatic signal
induced the yolk-sac stem cells to differentiate along the granulocytic pathway but did not
stimulate yolk-sac CFUs growth. Contrary to CFUs originating from foetal liver or adult bone
marrow, yolk-sac CFUs do not increase numerically in diffusion chamber culture.
INTRODUCTION
The current view of vertebrate haematopoietic ontogenesis holds that a succession of pluripotent stem-cell migrations originate in the yolk-sac blood islands
and invade the hepatic rudiment first and then the spleen and bone marrow
(Moore & Owen, 1967a,b; Moore & Metcalf, 1970). However, this view has
been challenged by other authors. Experimental evidence in birds (DieterlenLievre, 1975; Dieterlen-Lievre, Beaupain & Martin, 1976; Toivanen, Eskola &
Toivanen, 1976; Beaupain, Martin & Dieterlen-Lievre, 1979; Martin et al.,
1979) in amphibians (Hollyfield, 1966; Volpe & Turpen, 1977) and in mice
(Marks & Rifkind, 1972a,b; Cudennec, Thiery & Le Douarin, 1981) questions
the dependence of the intraembryonic haematopoiesis on the migratory stream
of haemopoietic stem cells which would develop ab initio within the yolk-sac
mesoderm.
We have previously studied the proliferation and the differentiation of visceral
and parietal yolk-sac cells from 9-day-old mouse embryos by the diffusion chamber (DC) technique (Symann etal., 1978). The yolk-sac blood islands are mainly
composed of primitive erythroblasts; macrophages are also occasionally present.
Between the 7th and 12th day of gestation there is no evidence of the production
1
Author's address: Laboratoire d'Hematologie Experimentale, Clos Chapelle-aux-Champs
30,2 U.C.L. 30.52, 1200 Bruxelles, Belgium.
Author's address: Ludwig Institute for Cancer Research (Brussels Branch), Avenue
Hippocrate 74, UCL 74.59, 1200 Bruxelles, Belgium.
88
M. A. ANCKAERT AND M. SYMANN
of other differentiated haemopoietic cell lines in the yolk-sac environment. In
the DC cultures of visceral yolk-sac cells CFUs were undetectable, macrophages
were the most numerous cells and granulocytes never exceed 6 % of all the
harvested cells. These findings gave results contrary to those obtained in the
foetal liver experiments (Symann et al. 1976a,b,c). Indeed, foetal liver CFUs
proliferate actively in DC. On the 16th gestational day, foetal liver, which is also
predominantly an erythroid organ, mainly generates vigorously growing
granulocytic cells and, to a lesser extent macrophages, in DC cultures.
The present study was designed to evaluate the hypothetical activity of foetal
hepatic factors on putative yolk-sac stem cells. For this purpose, we used the
double diffusion chamber (DDC) technique introduced by Pfeffer & Boyum
(1977). Briefly, the DDC were made of a regulator compartment, where a tissue
capable of producing a haematopoietic regulation factor was introduced and a
test compartment where a target population was cultured.
In DDC, a hepatic signal induced differentiation of the yolk-sac stem cells
along the granulocytic pathway. This differentiation did not occur in this signal's
absence.
MATERIALS AND METHODS
Animals
In all experiments virgin female C3H mice (Centre d'Animaux de
Laboratoire, Heverlee, Belgium) 10-16 weeks old were used as single diffusion
chamber (SDC) and double diffusion chamber (DDC) hosts or as irradiated
recipients of spleen colony assay. Yolk-sac donors were C3H mice mated overnight and sacrificed 9 days later. The stage reached by the embryo was carefully
checked by counting somite pairs: only the embryos which did not develop
beyond the 17-somite stage were retained. Yolk sacs were dissected as previously
described (Symann et al., 1978). Foetal liver donors were C3H mice mated
overnight and sacrificed 13 days later or WISTAR rats mated overnight and
sacrificed 13 days later. All animals were killed by cervical dislocation. All the
manipulations were performed on a laminar airflowbench at room temperature.
Single diffusion chamber culture
We used the single diffusion chamber (SDC) technique as described by Benestad (1970). Yolk-sac cells, obtained by passing the yolk sacs through needles of
sequentially decreasing diameters, were cultured. After 6 days in culture, the
SDC were removed from the host animal and incubated in Hanks Balanced Salt
Solution (HBSS) with 0-5 % pronase for 1 h at 25 °C. The cells were harvested
and counted in a haemocytometer.
Double diffusion chamber culture
We used the double diffusion chamber (DDC) technique as described by
Granulopoiesis in visceral yolk-sac cells
89
Pfeffer & Boyum (1977). The DDC consisted of a regulator compartment in
which five mouse foetal livers from 13-day-old foetuses or five rat foetal livers
from 13-day-old foetuses were introduced and a test compartment with murine
yolk-sac cells. After 6 days of culture, the DDC were treated as SDC and the
contents of the two different compartments harvested. The cells from each
compartment were counted in a haemocytometer, smeared and processed for
CFUs assays.
CFUs assay
Pluripotent stem cells were assayed according to the Till & McCulloch (1961)
method. 0-25 ml of cell suspension in HBSS was injected via the tail vein into
three to nine lethally irradiated (950R from a caesium source) mice. In these
experiments irradiated mice received, respectively, 1 x 106 visceral yolk-sac
cells and 1 x 106 DDC test compartment harvested cells. CFUs assays have been
performed at least twice with each kind of cell suspension.
Cytology
Smears of yolk-sac cells and foetal liver cells prior to and after diffusion
chamber culture were made using a cytocentrifuge (Shandon). The slides were
air dried, fixed with absolute methanol and then stained with May-GriinwaldGiemsa or with ortho-tolidine and counterstained with Giemsa (a modification
of the Sato method). In addition, other slides were stained by the sodium alphanaphtylphosphate according to the Gomori (Gabe, 1968) method to identify
alkaline phosphatases. Differential counts were based on the evaluation of at
least 300 cells per smear. The cells were classified as proliferative granulocytes
(myeloblasts, promyelocytes and myelocytes), non-proliferative granulocytes
(metamyelocytes, bands and segmented granulocytes) or macrophages. Differentiated erythroid cells were classified together as erythroblast.
Statistics
All the results are expressed as weighted mean and standard error of the mean
which were calculated as described by Armitage (1971).
RESULTS
1. Yolk-sac cells proliferation and differentiation in SDC and DDC
The yolk-sac cells were cultured in DC in four different ways: 300000 murine
vitelline cells were introduced into SDC, or into the test compartment of a DDC
where the regulator compartment was either empty or contained five 13-day
mouse or rat foetal livers. Murine yolk-sac cells were cultured in the presence of
rat foetal liver tissue to demonstrate that liver cells could not cross the Millipore
filter. Indeed, contrary to mouse neutrophils, rat neutrophils contain alkaline
90
M. A. ANCKAERT AND M. SYMANN
phosphatases. Thus, staining of the cell contents of each DDC compartment by
sodium alpha-naphtylphosphate allowed us to determine the cells' real source.
This checked our DDC system's cell tightness.
The presence of foetal hepatic tissue in the DDC regulator compartment did
not significantly (P = 0-05) influence the total cell production by the visceral yolk
sac present in the test compartment (Fig. 1). The small decrease of DDC cell
yield could not thus be attributed to the presence of the foetal hepatic tissue in
the regulator compartment since it was identical in the DDC with an empty
regulator compartment.
When foetal liver was present in the regulator compartment, granulocytic
proliferation was significantly increased at the expense of macrophage production. Figure 2 illustrates macrophage proliferation. The presence of foetal hepatic tissue reduced visceral yolk-sac cells macrophage production when compared
to results obtained from SDC (P< 0-05).
Figure 3 shows the production of non-proliferative granulocytes after 6 days
of culture. 6000 ± 1000 mature granulocytes were collected from SDC, 5000 ±
900 from DDC without foetal hepatic tissue, 19 000 ±3000 from DDC with
mouse foetal livers and 12 000 ± 3000 from DDC containing rat foetal tissue. The.
presence of mouse or rat foetal liver in the regulator compartment increased the
production of mature granulocytes in the test compartment when compared to
results obtained from SDC or DDC with an empty regulator compartment
200000—
P4
SDC (YS) DDC (YS/-) DDC (YS/FLM) DDC (YS/FLR)
(n = 7)
(n = 7)
(n = 9)
(n = 7)
w
5 150000—
II
100000—
CD
•
50000—
0—
DAY SIX OF DC CULTURE
Fig. 1. Total production of 9-day visceral yolk-sac cells in SDC and DDC culture
after 6 days. Each point is the weighted mean ± S.E.M. of pooled data from three
replicate experiments. The number of chamber is shown. SDC(YS): culture of yolksac cells in SDC. DDC(YS/-): culture of yolk-sac cells in DDC where the regulator
compartment was empty. DDC(YS/FLM): culture of yolk-sac cells in DDC where
the regulator compartment contained five mouse 13-day foetal liver. DDC(YS/
FLR): culture of yolk-sac cells in DDC where the regulator compartment contained
five rat 13-day foetal liver.
Granulopoiesis in visceral yolk-sac cells
91
SDC (YS) DDC (YS/-) DDC (YS/FLM) DDC (YS/FLR)
(n = 7)
(n = 7)
(n = 9)
(n = 7)
5 150000—
w
Cu
W
o
100000—
P
50000—
0—
DAY SIX OF DC CULTURE
Fig. 2. Macrophages production of 9-day visceral yolk sac in SDC and DDC culture
after 6 days. Each point is the weighted mean ± S.E.M. of pooled data from three
replicate experiments. The number of chamber is shown. Abbreviations as in Figure 1.
SDC (YS) DDC (YS/-) DDC (YS/FLM) DDC (YS/FLR)
(n = 7 )
(n = 7)
(n = 9)
(n = 7)
"a!
SI 20000
O
9 gW
15 000—
10000—
5 000—
0—
DAY SIX OF DC CULTURE
Fig. 3. Production of yolk-sac nonproliferative granulocytes after 6 days of SDC and
DDC culture. Each point is the weighted mean ± S.E.M. of pooled data from three
replicate experiments. The number of chamber is shown. Abbreviations as in Figure 1.
(P<0-05). This phenomenon was even more striking in the production of
proliferative granulocytes. There were 2500 ± 600 granulocytes in SDC, 1900 ±
600 in DDC without foetal hepatic tissue, 11000 ± 3000 in DDC withfivemouse
foetal livers and 10800 ± 1800 in DDC withfiverat foetal livers after 6 days of
culture (Fig. 4). Again the presence of foetal liver in the regulator compartment
enhanced the production of proliferative granulocytes in the test compartment
(P< 0-05 for SDC or DDC(YS/-) versus DDC(YS/FLM) or DDC(YS/FLR)).
The influence of the foetal hepatic environment upon the erythroid population
92
M . A . A N C K A E R T A N D M. S Y M A N N
SDC (YS) DDC (YS/-) DDC (YS/FLM) DDC (YS/FLR)
(n = 7)
(n = 7)
(n = 9)
(n = 7)
w « 15 000—
~ u 10000-
C
5 000—
i
0—
DAY SIX OF DC CULTURE
Fig. 4. Production of yolk-sac proliferative granulocytes after 6 days of SDC and
DDC culture. Each point is the weighted mean ± S.E.M. of pooled data from three
replicate experiments. The number of chamber is shown. Abbreviations as in Figure 1.
SDC (YS) DDC (YS/-) DDC (YS/FLM) DDC (YS/FLR)
(n = 7)
(n = 7)
(n = 9)
(n = 7)
20000
S S 15 000-
10000—
5 000—
0—
DAY SIX OF DC CULTURE
Fig. 5. Production of yolk-sac nucleated erythrocytes in SDC and DDC culture.
Each point is the weighted mean ± S.E.M. of pooled data from three replicate experiments. The number of chamber is shown. Abbreviations as in Figure 1.
is shown in Fig. 5. No statistic difference between the various DC culture was
observed.
2. CFUs assay
In order to check whether the yolk sac contained pluripotent stem cells and
whether the foetal hepatic tissue was able to influence their proliferation, a
CFUs assay was performed after dissection and after SDC and DDC cultures in
the presence of 13-day foetal livers. CFUs were almost undetectable after yolksac dissection or after conventional DC culture if 1 x 106 cells were injected into
Granulopoiesis
in visceral yolk-sac cells
93
lethally irradiated mice. A similar result was obtained after DDC culture with
foetal liver tissue in the regulator compartment (Table 1).
DISCUSSION
Our experiments provide evidence that the properties of vitelline haemopoietic stem cells are different from those of foetal liver CFUs or adult bone
marrow CFUs. It should be stressed that their detection by the spleen colony
assay is questionnable (Table 1). Like Niewish et al. (1970) we did not find any
significant level of visceral yolk-sac CFUs in the various experimental DC system
situations. According to Moore & Metcalf (1970) and to our previous results
(Symann et al., 1978) the incidence of CFUs in the yolk sac is very low. Perah
& Feldman (1977) claimed that after 48h in vitro, undetectable yolk-sac CFUs
become activated and may increase as much as 84-fold. The 6-day DC culture did
not permit activation of day-9 yolk-sac stem cells nor did a humoral influence
from day-13 foetal hepatic tissue in DDC culture (Table 1). Contrary to yolk-sac
CFUs, foetal liver CFUs proliferate and increase numerically in DC culture even
faster than adult bone-marrow CFUs (Symann etal., 1976a,c; Breivik, Benestad
&B6yum, 1971).
When the vitelline cells were submitted to the influence of a hepatic environment by the DDC culture, a hepatic signal from the day-13 foetal liver induced
the yolk-sac stem cells to differentiate along the granulocytic pathway but did not
stimulate yolk-sac CFUs to increase in number. Indeed, granulocytic production
increased more than four-fold when the foetal hepatic tissue was introduced in
the DDC regulator compartment (Figs 3, 4). Cudennec et al. (1978) have also
documented the influence of hepatic factors upon yolk-sac haemopoiesis and
have analysed the in vitro capacity of yolk-sac haemopoietic cells to produce
either primitive or definitive erythrocytes. Prior to the colonization of the liver
rudiment by haemopoietic cells, yolk sac explanted alone produced solely
primitive erythrocytes and only for a short time. When allowed to colonize a liver
Table 1. CFUs assay from 9-day visceral yolk sac before and after DDC cultures
in presence of 13-day foetal livers
Cell suspension
Number of spleen
colonies
Saline
Y.S.
DDC (Y.S./-)
DDC(Y.S./FLM)
0-44 ± 0-24
1-25 ±0-63
0
0-2 ±0-2
Results are expressed as mean ±S.E.M. of pooled data from at least two different experiments. In each experiment, mice were injected i.v. with 1 x 106 cells in 0-25 ml of HBSS.
EMB73
94
M. A. ANCKAERT AND M. SYMANN
rudiment, haemopoietic cells from the yolk sac gave rise to definitive erythrocytes. These cells could express the same capacity when stimulated by liver
rudiment even if no direct cell-cell contact was established between stimulating
tissue and target haemopoietic cells.
Murine foetal liver is predominantly an erythropoietic organ enclosing few
granulocytic cells. Foetal haemopoietic stem cells do not seem to express their
granulocytic potential in the foetal liver microenvironment. However, the foetal
liver cell suspensions as well as the adult bone-marrow cells generate almost
exclusively granulocytes and to a lesser extend macrophages in diffusion chamber cultures (Symann et al., 19766; Vilpo & Vilpo, 1976). This suggests that the
induction of the process of differentiation in our experiments with DDC results
from a foetal liver signal, while the choice of a specific pathway, the granulocytic
expression in this instance, comes from the diffusion chamber's microenvironment.
To summarize, our results show that yolk-sac CFUs have different properties
than CFUs originating from foetal liver or adult bone marrow. In DDC, a hepatic
signal induced the yolk-sac stem cells to the granulocytic differentiation.
RESUME
Par la technique de culture en doubles chambres a diffusion (DDC), nous
avons cherche a evaluer l'influence eventuelle de facteurs hepatiques foetaux sur
de putatives cellules souches vitellines. La DDC comprend un compartiment
regulateur ou est introduit du tissu hepatique foetal et un compartiment test ou
les cellules du sac vitellin visceral sont cultivees. Dans ce systeme, un signal
hepatique oriente les cellules souches vitellines vers la differentiation
granulocytaire mais n'induit pas une augmentation numerique des CFUs
vitellines. Contrairement aux CFUs provenant du foie foetal ou de la moelle
osseuse adulte, les CFUs du sac vitellin n'augmentent pas en nombre lorsqu'elles
sont cultivees en chambres a diffusion.
We are indebted to Drs J. Rodhain and A. Fedida for their help. We gratefully acknowledge
Mrs D. Chintinne for her technical assistance and Mrs K. Deleuse for the expert typing of the
manuscript.
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