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.

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 and in ectopic sites (Rossant, 1977: Rossant & Ofer, 1977). It is not clear whether the effect of 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.

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)4·10% foetal calf serum (FCS) and a-modified MEM (Gibco)4-10% 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 α-MEM4-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 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 μm 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 α-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 μ g/ml of colcemid for 2 h before cell spreads were prepared and scored 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.

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).

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 by fibroblastic-type cells, probably of mesodermal origin.

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 by fibroblastic-type cells, probably of mesodermal origin.

Cell division and ploidy 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.

Table 1

Mitotic indices of embryonic fragments, EE and EPC under different culture conditions

Mitotic indices of embryonic fragments, EE and EPC under different culture conditions
Mitotic indices of embryonic fragments, EE and EPC under different culture conditions

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 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.

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.

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.

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. 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.

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.

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).

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. 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.

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).

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).

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.

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.

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 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 maintenance 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-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 blastocyst 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.

Fig. 7

Proposed model for the postimplantation trophoblast cell lineage in the mouse.

Fig. 7

Proposed model for the postimplantation trophoblast cell lineage in the mouse.

This work was supported by the Canadian Natural Sciences and Engineering Research Council.

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