/ . Embryol. exp. Morph. Vol. 62, pp. 217-227, 1981
Printed in Great Britain © Company of Biologists Limited 1981
217
Effect of culture conditions
on diploid to giant-cell transformation in
postimplantation mouse trophoblast
By J. ROSSANT 1 AND W. TAMURA-LIS 2
From the Department of Biological Sciences, Brock University,
Ontario
SUMMARY
Diploid extraembryonic ectoderm and ectoplacental cone from the 7-5-day mouse embryo
were grown in vitro under a variety of culture conditions in an attempt to discover conditions
which maintain trophoblast in a diploid state and prevent giant-cell formation. It was found
that maintenance of tissue integrity was not enough to keep the tissues dividing and diploid,
but that the presence of inner-cell-mass derivatives did have some effect. This effect was only
apparent when trophoblast cells were entirely enclosed by embryonic tissues. Monolayers of
embryonic or embryonal carcinoma cells did not prevent giant-cell formation. Diploid
extraembryonic ectoderm and ectoplacental cone responded differently: ectoplacental cells
eventually formed trophoblast giant cells even when enclosed by embryonic cells
whereas extraembryonic ectoderm cells apparently could be maintained in a diploid condition. This and other differences in properties between extraembryonic ectoderm and
ectoplacental cone are discussed with reference to a new model for the postimplantation
trophoblast lineage in the mouse.
INTRODUCTION
Trophoblast proliferation at the late blastocyst stage in the mouse depends on
the presence of the inner cell mass (ICM). Trophectoderm cells that are in
contact with the ICM continue to divide and remain diploid, while cells that
move away from the ICM cease division and start to endoreduplicate their DNA
to form trophoblast giant cells (Gardner, 1972; Barlow & Sherman, 1972;
Gardner, Papaioannou & Barton, 1973; Ansell & Snow, 1975; Copp, 1978).
There is also evidence that the continued presence of ICM derivatives is required
to maintain diploidy in postimplantation trophoblast (Gardner & Papaioannou,
1975; Rossant, 1977). Secondary giant-cell transformation only occurs in vivo in
the part of the ectoplacental cone furthest from the embryonic region and
separation of diploid trophoblast tissues from ICM derivatives as late as 8-5 or
9-5 days of development leads to giant-cell transformation in vitro andinectopic
sites (Rossant, 1977: Rossant & Ofer, 1977). It is not clear whether the effect of
1
Author's address: Department of Biological Sciences, Brock University, St Catharines,
Ontario,
L2S 3A1, Canada.
a
Author's address: Department of Biology, Illinois Institute of Technology, Chicago,
Illinois, 60616, U.S.A.
218
J. ROSSANT AND W. TAMURA-LIS
the ICM derivatives is mediated by a specific inductive stimulus or by physically
maintaining the close cell contacts and tissue organization of the dividing
trophoblast (Rossant, 1977), since previous experiments showing giant-cell
transformation in isolated diploid trophoblast have always involved disruption
of normal tissue organization (Rossant & Ofer, 1977). In the present study, we
have grown diploid trophoblast tissues under culture conditions where tissue integrity is maintained and compared their ploidy and proliferation with those of
the same tissues enclosed by or in contact with embryonic cells. We find no
evidence that maintaining close cell contacts per se will keep trophoblast in a
diploid state, whereas enclosing trophoblast loosely in embryonic tissues does
have some effect on maintaining diploidy, suggesting that ICM derivatives may
exert a specific inductive effect.
MATERIALS AND METHODS
Recovery and dissection of embryos
All mice used were from a closed stock of originally random bred Ha(ICR)
mice (West Seneca Breeding Facility, Roswell Park Memorial Institute, Buffalo,
N.Y.). PB1 medium (Whittingham & Wales, 1969)+ 10% foetal calf serum
(FCS) and a-modified MEM (Gibco) + 10% FCS were used for recovery, dissection and transfer of embryonic tissues. Embryos were dissected from the
uteri of mice on the afternoon of the eighth day after natural mating (7-5-day
embryos). Diploid ectoplacental cone (EPC) and extraembryonic ectoderm (EE)
were separated from the embryos by dissection and enzyme treatment as
described previously (Johnson & Rossant, 1980). Little or no contamination
with giant cells was observed by microdensitometry (Johnson & Rossant, 1980).
EE fragments were usually larger than EPCs and so were cut in half using
glass microneedles, so that all fragments were approximately the same size
(c. 1000-1500 cells in air-dried spreads). The embryonic regions enclosed by
the amnion were not treated with enzyme but were used as 'pockets' for culture
of EE and EPC.
Culture and ectopic transfer of tissues
Pieces of EE and EPC were subjected to a variety of different culture conditions.
1. EPC and EE were grown in suspension in a-MEM + FCS in bacteriological
culture dishes.
2. EPC and EE were grown in a collagen lattice suspended in a-MEM
(Elsdale & Bard, 1972). Five ml of the collagen mixture was poured into 60 mm
culture dishes and pieces of EPC or EE were suspended in the lattice just before
it set. Care was taken to avoid mechanical agitation of the dishes since this
destroys the gel.
3. Single pieces of EPC or EE were inserted inside the amniotic cavities of
Giant-cell transformation in mouse trophoblast
219
intact embryonic fragments before further culture in bacteriological dishes.
Some embryonic fragments containing EE or EPC were also transferred beneath
the testis capsules of male mice. After 7 days, the recipients were killed and
examined for haemorrhagic graft sites. All testes were fixed, embedded, and
sectioned at 7 /im so that the graft sites could be examined microscopically.
4. Monolayers of embryonic cells were prepared by Trypsin treatment of
9-5-day embryos (EPC and EE excluded).The cells were plated out at a concentration of 105 cells/well in Microtest plates (Falcon, Microtest II) and
allowed to attach for a few hours before each well was seeded with a single piece
of EE or EPC.
5. EE and EPC were grown on spreads of embryonal carcinoma (EC) cells.
P19 cells (a pluripotent line of EC cells originally derived from a C3H embryo
and obtained from Dr M. W. McBurney, University of Ottawa) were grown
until almost confluent in a-MEM + FCS and then seeded with EE or EPC.
All cultures (1-5) were gassed with 5 % CO2 in air and maintained in a humidified atmosphere at 37 °C.
Mitotic index measurement and microdensitometry
The mitotic activity of EE and EPC was assessed after 24 or 48 h under the
various culture conditions. EE and EPC could not be readily recognized and
dissected out of embryonic pockets later than 2 days of culture and so full
comparisons could not be made beyond 48 h. All cultures were treated with
1 /*g/ml of colcemid for 2 h before cell spreads were prepared and icored blind
for metaphases as described previously (Rossant & Ofer, 1977).
Some cell spreads were Feulgen-stained for microdensitometric determination
of DNA levels (Pearse, 1972). Microdensitometry was performed using a Leitz
MPV microspectrophotometer as described previously (Johnson & Rossant,
1980). Control liver cell spreads were used to calibrate the histograms obtained.
RESULTS
Trophoblast growth under different culture conditions
EE and EPC tissues appeared healthy under all culture conditions and were
capable of uptake and incorporation of [3H]thymidine (unpublished data).
Fragments grown in suspension or in collagen gels remained as discrete lumps
whereas fragments grown on embryonic or EC cells attached to and invaded the
underlying cells. EE and EPC inside embryonic 'pockets' also remained as
discrete lumps which were only loosely attached to the surrounding embryonic
tissues (Fig. 1).
220
J. ROSSANT AND W. TAMURA-LIS
.*)
Fig. 1. Section of EE in embryonic pocket after 2 days of culture. EE cells remain as
a solid lump and appear to be loosely surrounded byfibroblastic-typecells, probably
of mesodermal origin.
Cell division andploidy in EE and EPC under different culture conditions
Culture conditions 1-3
Previous studies have shown that EE and EPC rapidly cease cell division when
isolated and grown in explant culture and later begin to endoreduplicate their
DNA (Rossant & Ofer, 1977; Johnson & Rossant, 1980). Comparison of the
mitotic indices (M.I.s) of EE and EPC under different culture conditions should,
therefore, give some indication of whether the diploid, proliferative state is being
maintained. The M.I.s of EE and EPC dropped drastically after 24 h in suspension or collagen gel cultures and by 2 days the M.I. of EE was only 9 % of its
initial value and mitotic figures were practically non-existent in the EPC cultures
(Table 1). The M.I.s of EE and EPC inside embryonic pockets were significantly
higher than in the other culture conditions at both 1 and 2 days of culture,
declining to 27 % of the initial M.I. for EE and 45 % of the initial value for EPC
by 2 days. The M.I. of the embryonic fragments enclosing the EE and EPC also
declined to a value of 31 % of the original M.I. by 2 days, suggesting that much
of the decline in M.I. of EE and EPC in embryonic pockets could be attributed
to a general decline in cell division in culture.
Confirmation of maintenance of diploidy in EE and EPC inside embryonic
pockets was sought using microdensitometric analysis of DNA levels but the
results were not clear-cut. EPC tissues contained some cells with DNA values
Giant-cell transformation in mouse
221
trophoblast
Table 1. Mitotic indices of embryonic fragments, EE and EPC under
different culture conditions
Type of
tissue
Embryonic
fragment
Initial M.I. + s.E.
(N)
13-9 + 0-80
(5)
Type of culture
condition
Suspension
Collagen gel
EE*
10-8 + 1-11
(5)
Suspension
Collagen gel
Embryonic
pocket
EPCt
5-6 + 0-82
(5)
Suspension
Collagen gel
Embryonic
pocket
M.I. ± S.E. after M.I. ± S.E. after
1 day (N)
2 days (N)
4-9 ±0-26
(22)
7-2 + 0-37
(11)
20 ±0-26
(12)
1-4 ±0-33
(11)
4-8 ±0-28
(12)
4-3 ±0-26
(19)
5-4 + 0-33
0-9±016
(12)
0-4±0-16
(12)
2-9 ±0-50
(12)
0-4 ±0-20
(12)
0-2 ±007
(10)
2-5 ±0-45
(13)
008 ±004
(12)
002 ±002
(7)
2-5 ±0-48
(12)
01)
* EE, Day 1.
F = 39-66
D.F. = 2,32
P < 001
EE, Day 2.
F = 17-54
D.F. = 2,33
P < 001
Scheflte's test (Guenther, 1964) revealed significant difference (P < 001) between M.I. of EE
in embryonic pocket and M.I. of EE in both suspension and collagen gels.
t EPC, Day 1.
F = 31-73
D.F. = 2,32
P < 001
EPC, Day 2.
F = 20-92
D.F. = 2,28
P < 001
Scheff6's test revealed significant difference (P < 001) between M.I. of EPC in embryonic
pocket and M.I. of EPC in both suspension and collagen gels.
greater than 4C in all three culture conditions (Fig. 2), despite the evidence for
continued proliferation of some EPC cells inside embryonic pockets. EE cells, on
the other hand, were mostly still diploid after 2 days in all three culture conditions, despite differing M.I.s (Fig. 2). However, it has previously been shown
that, although isolated EE cells cease division fairly rapidly, they do not begin to
endoreduplicate their DNA until 3-4 days of explant culture (Johnson &
Rossant, 1980). Indeed, continued culture beyond 2 days of EE in suspension or
collagen gels did result in giant-cell formation (data not shown) but EE in
embryonic pockets could not be isolated after 2 days to determine whether cells
remained diploid. However, examination of ectopic grafts suggested that this was
the case. All ten grafts of EPC inside embryonic pockets contained proliferating
embryonic tissues enclosing a haemorrhagic area with trophoblast giant cells
(Fig. 3). However, all six grafts of EE inside embryonic pockets showed only
embryonic-type tissues: no haemorrhage or giant cells could be found (Fig. 4).
All control grafts of EE (N = 8) and EPC (N = 11) alone produced haemorrhagic grafts with giant cells.
8
EMB 62
222
J. ROSSANT AND W. TAMURA-LIS
2C
8C
4C
EEs
2C
4C
8C
i
EPCs
150
200
8C
r-H.
50
2C
EEc
2 5
20
100
4C
50
2C
r
A
100
4C
150
8C
|
EEe
200
-
• —
50
V
P-lJ
i
150
100
Absorbance units
8C
100
4C
150
200
250
50
300
8C
i
i
EPCe
n
i
200
i
300
I5
50
2C
n
- •
n
250
EPCc
i
5
200
§10
15 10
150
100
150
200
250
n.300
Absorbance units
Fig. 2. Histograms of DNA values of EE and EPC after 2 days in different culture
conditions. C values taken from control liver cell readings (data not shown), s, Suspension culture, c, Collagen culture, e, Culture inside embryonic pocket.
Culture conditions 4 and 5
Results from conditions 1-3 suggested that embryonic tissues could maintain
diploidy at least in EE tissues, but the difficulty of isolating trophoblast beyond
48 h in culture hindered further analysis. In an attempt to overcome this problem, EE and EPC were explanted on monolayers of 9-5-day embryonic cells.
Only a small series of such experiments were carried out because it was clear that
both EE and EPC invaded the confluent monolayer and transformed into giant
cells after 2-3 days of culture (Fig. 5). Mitotic activity was almost completely
absent in both EE and EPC after 2 days (data not shown).
It is possible that the 9-5-day embryo cells used to make monolayers were no
longer competent to maintain diploidy in trophoblast. However, use of earlier
embryos would be very difficult since cell numbers are low and many embryos
would be needed to make one small monolayer culture. No such restrictions
apply to the use of EC cells. These teratocarcinoma cells are often considered
analogous to ICM or early uncommitted ICM derivatives (Martin, 1975) and so
might be capable of maintaining trophoblast proliferation (Papaioannou, 1979).
Giant-cell transformation in mouse trophoblast
223
50 M m
Fig. 3. Representative section of EPC in embryonic pocket grafted under the testis
capsule. Giant cells (GC) and haemorrhage are enclosed by embryonic cells (E).
Fig. 4. Representative section of EE in embryonic pocket grafted under the testis capsule. No giant cells or haemorrhage are visible: only a variety of embryonic tissues
are found.
However, EE and EPC cells also invaded EC cultures., pushing the EC cells
aside (Fig. 6). After 2 days culture in the presence of EC cells the M.I. of EE
was low (1-34 ±0-23, N = 5) and no mitotic figures could be detected in the
EPC cultures (N = 10). By 3 days of culture, giant cells were clearly visible in
both EE and EPC spreads, often in close proximity to EC cells (Fig. 6).
DISCUSSION
Although some trophoblast cells remain diploid and continue to divide
throughout a large part of mouse postimplantation development, it has proved
difficult to maintain trophoblast proliferation in vitro. Simply preserving tissue
8-2
J. ROSSANT AND W. TAMURA-LIS
50 pim
Fig. 5. EE outgrowth on 9-5-day embryonic cells after 3 days in culture. EE cells (on
left) are spreading and displacing embryonic cells (on right). Giant cells are visible at
the edge of the EE outgrowth.
Fig. 6. EPC outgrowth on EC cells after 3 days in culture. EPC cells (on right) are
spreading and displacing EC cells (at upper left). Nearly all EPC cells appear giant.
organization and integrity by growing tissues either in suspension culture or in
collagen lattices did not maintain diploidy in either EE or EPC. The mitotic
indices and DNA values of the two tissues were very similar to those obtained in
explant cultures (Rossant & Ofer, 1977; Johnson & Rossant, 1980). However,
both EE and EPC showed significantly higher mitotic indices when enclosed by
embryonic tissues. Indeed EE cells may remain diploid for some time when
enclosed by embryonic tissue, since ectopic grafts of EE in embryonic pockets
showed no sign of giant cell transformation after 7 days. It was not possible to
prove that the EE cells were still present in the grafts without a cell marker but
this seemed likely since EE alone did survive and produce giant cells under the
testis capsule. EPC cells, on the other hand, did not show such complete main-
Giant-cell transformation in mouse trophoblast
225
Inner cell
mass
|
Mural primary
giant cells
Morula—»• Trophectoderm—»• Polar
—•- Extraembryonic —*• Diploid
I
I trophectoderm I ectoderm
ectoplacental
2-5 days
35 days
I
4-5 days
»• Secondary
giant cells
Post implantation
Fig. 7. Proposed model for the postimplantation trophoblast cell lineage in the
mouse.
tenance of diploidy when enclosed by embryonic cells. Although the mitotic
index of EPC in embryonic pockets was still quite high after 2 days in culture,
microdensitometry revealed that some cells had actually undergone endoreduplication and ectopic grafts of EPC in embryonic pockets all showed extensive
giant-cell formation. Monolayers of either embryonic or embryonal carcinoma
cells were not effective in preventing giant cell formation in either EE or EPC.
Thus, of all the different culture conditions tested, only enclosing EE in embryonic tissues produced any effect on preventing giant-cell formation, suggesting
that the geometry of interaction between EE and embryonic cells may be
important. Further study will be required to determine whether this results from
an accumulation of a diffusible inductive substance inside the embryonic pockets
or from some effect on tissue mass or tissue architecture other than simply
maintaining close trophoblast cell-cell contact.
Our failure to prevent eventual giant cell formation by EPC under any
culture conditions indicates that EE and EPC do not possess identical properties, although they are both diploid trophectoderm derivatives. Previous work
has revealed other differences between EE and EPC (Johnson & Rossant, 1980).
The protein synthetic patterns of the two tissues differed, as judged by 2-D gel
electrophoresis, and during in vitro culture EE cells apparently passed through a
brief stage of remaining diploid and synthesizing EPC-like proteins before
transforming into giant cells. EPC cells rapidly commenced giant-cell transformation. These observations lead us to propose a model for the postimplantation trophoblast lineage in which EE cells act as stem cells for all other tropho-
226
J. ROSSANT AND W. TAMURA-LIS
blast cell types (Fig. 7). Copp (1979) has proposed a morphogenetic model for
trophoblast formation in the early egg cylinder which suggests that EE is the
first tissue to be formed from the polar trophectoderm and that EPC is formed
later as mechanical constraints force the trophoblast to grow outwards into the
uterine crypt. Our present model extends this hypothesis into the later
embryo, and proposes that the morphogenetic sequence reflects underlying
differentiative events. A unidirectional pathway of differentiation is envisaged,
in which the diploid polar trophectoderm of the blastocyst gives rise to the
extraembryonic ectoderm. This tissue is then capable of continued self-renewal
or of producing diploid ectoplacental cone cells. EPC cells have only a limited
capacity for self-renewal and are committed to secondary giant-cell formation.
EE cells cannot give rise directly to giant cells and EPC cells cannot revert to EE.
The model should be testable by blastccyst injection (Rossant, Gardner &
Alexandre, 1978), where one would predict that injected EE should be capable
of colonizing EE, EPC and GC while EPC should only colonize GC, with
perhaps a minor contribution to EPC. Such experiments are under way.
This work was supported by the Canadian Natural Sciences and Engineering Research
Council.
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