J. Embryol. exp. Morph. Vol. 70, pp. 99-112, 1982
99
Printed in Great Britain © Company of Biologists Limited 1982
The developmental potential of a
euploid male teratocarcinoma cell line after
blastocyst injection
By J. ROSSANT 1 AND M. W. M C B U R N E Y 2
From the Department of Biological Sciences, Brock University,
and the
Departments of Biology and Medicine, University of Ottawa
SUMMARY
A karyotypically normal male embryonal carcinoma (EC) cell line, PI 9, produced large
numbers of chimaeras in midgestation after groups of cells were injected into mouse blastocysts. A wide variety of apparently normal tissues were colonized by the EC cells but most
chimaeras were also morphologically abnormal. Few live chimaeras were produced and all
contained tumours of EC cell origin as well as EC contributions to normal tissues. This
apparently incomplete regulation of the EC cells by the embryonic environment was not due
to EC cell variation, since a clonal subline, P19S18, produced similar patterns of colonization.
It was also not caused by the inability of the blastocyst to regulate large numbers of injected
EC cells, since a single P19S18 cell could contribute to both normal and tumour tissue in
the same mouse. Neither a high rate of colonization of the embryo nor a normal karyotype is,
therefore, sufficient to ensure reversion of EC cells to normal embryonic behaviour.
INTRODUCTION
Murine teratocarcinomas are malignant tumours which arise spontaneously
in the gonads of certain strains of mice (Stevens, 1973; Stevens & Varnum, 1974)
and can be induced experimentally by grafting early embryos (Stevens, 1970#)
or foetal genital ridges (Stevens, 19706) to ectopic sites. These tumours contain
a variety of differentiated cell types as well as an undifferentiated stem cell
population, known as embryonal carcinoma (EC). EC cells can be isolated
from the tumours and maintained in an undifferentiated form in culture
(reviewed Martin, 1975; Graham, 1977). Under suitable culture conditions
(Martin & Evans, 1975; Nicolas et al. 1976; McBurney, 1976) or after return
to ectopic sites in vivo (Kleinsmith & Pierce, 1964), EC cells can differentiate
into virtually all cell types observed in the original tumour. EC cells thus
1
Author's address: Dept. of Biological Sciences, Brock University, St. Catharines, Ontario
L2S 3A1, Canada.
2
Author's address: Depts. of Biology and Medicine, University of Ottawa, Ottawa,
Ontario KIN 6N5, Canada.
100
J. ROSSANT AND M. W. MCBURNEY
resemble cells of the early embryo in their pluripotency and they also share
biochemical, morphological and antigenic properties with pluripotent embryonic
cells (reviewed Graham, 1977). The ability to grow large numbers of EC cells
in vitro and study their differentiation has made the murine teratocarcinoma a
popular model system for investigating normal embryonic development
(Martin, 1975, 1980).
The validity of this model system has been reinforced by the discovery that
EC cells can form chimaeric mice after blastocyst injection (Brinster, 1974;
Mintz & Illmensee, 1975; Papaioannou, McBurney, Gardner & Evans, 1975;
Illmensee & Mintz, 1976; Dewey, Martin, Martin & Mintz, 1977). Both in vivo
and in vitro cell lines have been tested and the mitotic descendents of the EC
cells have been reported to contribute to a wide variety of normal tissues (Mintz
& Illmensee, 1975; Mintz & Cronmiller, 1978). However, until recently, only
EC cells from in vivo tumours, which are apparently euploid, have been shown
to contribute to all somatic tissues and to the germline (Mintz & Illmensee,
1975; Illmensee, 1978; Cronmiller & Mintz, 1978). In vitro cell lines have
mostly produced low numbers of chimaeras with limited somatic tissue colonization (Dewey et al. 1977; Papiaoannou et al. 1978). Several lines have also
produced tumours in adult chimaeric mice (reviewed Papaioannou, 1980),
showing that reversion of EC cells to normal embryonic behaviour is not
always complete following blastocyst injection. Most in vitro cell lines used
have shown varying degrees of aneuploidy, which could explain some or all
of the differences between the potential of in vitro and in vivo EC cells. In
particular, aneuploid cells are unlikely to be able to undergo normal meiosis
and form functional germ cells. It is important, therefore, to derive euploid
in vitro EC cell lines and compare their potential with normal embryonic cells
and with in vivo teratocarcinomas. Normal embryonic differentiation of such
cells following blastocyst injection would help to validate the use of the teratocarcinoma as a model system for studying embryogenesis and might also further
the aim of using EC cell lines to introduce new mutant genes into the mouse
germ line (Dewey et al. 1977). The developmental potential of three apparently
euploid cell lines was assessed by Papaioannou and her co-workers
(Papaioannou, Evans, Gardner & Graham, 1979; Papaioannou, 1980). Only
one line (C145b) formed any chimaeras and, in these, mosaicism was restricted
to yolk sac tissues. Mintz, however, has recently described a new female euploid
EC cell line, METT-1 (Mintz & Cronmiller, 1981), which formed chimaeras
after injection of 3-5 cells into blastocysts (Stewart & Mintz, 1981). Mosaicism
was widespread in somatic tissues and one chimaera also showed germ-line
mosaicism. A karyotypically normal male cell line (P19) has recently been
isolated in one of our laboratories (McBurney & Rogers, 1982) and we describe
here its developmental potential following blastocyst injection.
Potential of a euploid male teratocarcinoma cell line
101
MATERIALS AND METHODS
Cell Lines
The EC cell line, PI9, was isolated from the primary tumour induced by a
7-5-day C3H/He embryo grafted to an adult testis (McBurney & Rogers, 1982).
The cells have a normal male karyotype. Cells of the subclone, P19S18, behave
in culture like the parental PI9 cells (Jones-Villeneuve, McBurney, Rogers, &
Kalnins, 1982). Both cell lines differentiate poorly under normal culture
conditions but extensive differentiation can be induced by chemical agents such
as retinoic acid (Jones-Villeneuve et al. 1982). Both cell lines were feederindependent and were maintained in a-modified MEM (Gibco) + 5% foetal
calf serum (FCS) + 5 % new-born calf serum (NBCS) by subculturing every two
days. No culture was maintained for longer than 4-6 weeks before being replaced
by newly thawed cells from frozen stocks of the original cell lines. This procedure was adopted to eliminate the possibility of progressive changes in EC
cell properties and karyotype with prolonged culture (Papaioannou et al. 1979).
Mice
Host blastocysts were obtained following natural mating of a stock of
originally random-bred HA(ICR) mice maintained at Brock University. This
stock was homozygous for the a allele of the glucose phosphate isomerase
(GPI) gene {Gpi-\a/Gpi-\a) whereas the cell lines were Gpi-lb/Gpi-\b, since they
were derived from C3H mice. The cell lines were also presumed to carry the
agouti pigment marker, whereas the HA(ICR) mice were albino, non-agouti.
The same stock of HA(ICR) mice was used to provide recipients for embryo
transfer.
Blastocyst injections
Blastocysts were obtained by flushing the uteri of HA(ICR) mice on the
afternoon of the 4th day after natural mating. Blastocysts were flushed using
PB1 medium (Whittingham & Wales, 1969) + 5% FCS + 5% NBCS and all
subsequent manipulations and transfers were performed in this medium.
EC cell aggregates were obtained by trypsinizing a sub-confluent culture of EC
cells and replating the culture in a bacteriological petri dish. Five to eight hours
later, aggregates that resembled ICMs in size and morphology were selected
and transferred to PB1 medium prior to blastocyst injection. Cell numbers in
such aggregates were scored from air-dried cell spreads (Tarkowski, 1966).
Single cells were obtained by trypsinizing sub-confluent EC cultures and
resuspending in PB1 medium. Cells were allowed to recover from trypsinization
for at least 1 h before injection. Both aggregates and single cells were injected
into the blastocoelic cavities of host blastocysts using a Leitz micromanipulator
102
J. ROSSANT AND M. W. MCBURNEY
assembly (Gardner, 1978). Injected blastocysts were allowed to recover for 1 h
before transfer to the uterine horns of HA(ICR) females on the 3rd day of
pseudopregnancy.
Analysis of development of injected blastocysts
Some recipients carrying injected blastocysts were killed at various stages of
pregnancy while others were allowed to go through to term. Conceptuses from
mid-term pregnancies were dissected from the uterus and prepared for GPI
analysis (Peterson, Frair & Wong, 1978; Rossant & Lis, 1979). At 9-5 days of
pregnancy, conceptuses were dissected into embryonic, yolk sac and trophoblast (ectoplacental cone and giant cells) fractions only. At later stages of
pregnancy, the placenta and yolk sac were retained as separate fractions but the
foetus itself was further dissected into its various component organs. Foetuses
older than 11-5 days were also scored for the presence of eye pigmentation.
Any gross abnormalities of development were noted.
Any live offspring delivered at term were observed carefully and allowed to
develop at least until weaning, unless abnormalities made survival doubtful.
All live offspring were scored for the presence of coat or eye pigmentation. At
autopsy, various organs and tissues were dissected from the mice for GPI
analysis.
Analysis of tumours
Preterm conceptuses and live young were carefully observed for the presence
of any tumour-like growths and the position and size of such growths were
noted. Wherever there was enough tissue, part of each growth was frozen for
GPI analysis and part was fixed for histological examination. Fixed material
was embedded in paraffin wax, serially sectioned and stained with haematoxylin
and eosin. When little tissue was available, only GPI analysis was performed.
RESULTS
Contribution of EC cell progeny to developing embryos
Both PI9 and P19S18 colonized the host blastocyst and formed chimaeric
embryos as judged by GPI analysis at mid-gestation and at term. The results of
all experiments are summarized in Table 1, where it can be seen that the rate of
chimaera production in mid-gestation was high, especially when large numbers
of cells (15-25) were injected. However, few live chimaeras were born and all of
these and many mid-term foetuses showed various morphological abnormalities. A detailed examination of results from each cell line is given below.
Potential of a euploid male teratocarcinoma cell line
103
Table 1. Summary of results of injecting P19 and P19S18 celts into blastocysts
No. live
embryos No. live
blastocysts No. con- live chimaeric embryos
abnormal
injected ceptuses embryos
(%)
No.
No.
Cell
line
No. cells
injected
Stage
analysed
P19
15-25
15-25
15-25
9-5 days
12-5 days
Term
49
109
20
37
101
—
37
30
20
21 (57)
11 (37)
1 (5)
14
9
1
P19S18
15-25
15-25
15-25
9-5-10-5 days
12-5 days
Term
39
10
42
36
8
—
32
6
15
22 (69)
2(33)
4(27)
12
2
4
P19S18
1
1
12-5-15-5 days
Term
200
36
150
—
135
20
20 (15)
1 (5)
16
1
Chimaeras produced by injection ofP19 aggregates into blastocysts
At 9-5 days of development, 57 % of live embryos were chimaeric as determined by GPI analysis (Table 2). Some chimaeric foetuses contained as much as
60 % P19 GPI isozyme. However, many of these embryos were grossly abnormal.
Abnormalities ranged from complete absence of organized embryonic tissue,
through abnormalities of neural tube closure, to apparently normal but retarded
development. Seven chimaeras were not obviously abnormal.
When injected embryos were allowed to proceed to 12-5 days, a large number
were resorbed (Table 1). All 71 resorptions were analyzed by GPI and 40
showed evidence of EC cell contribution. When these figures are added to the
11 chimaeras found among the live foetuses, the overall rate of chimaerism is
50%. Ten of the eleven live chimaeras showed eye pigment mosaicism as well
as EC cell contribution to various internal tissues (Table 3). However, eight of
these and one further chimaera without eye pigment were morphologically
abnormal. The head region showed the most obvious abnormalities, ranging
from complete failure of neural tube closure to patches of loose, excess skin on
the snout.
In one experiment, the injected embryos were allowed to go through to term
(Table 1), and in this particular experiment, all foetuses survived. Only one
mouse was chimaeric and this mouse was killed at 2 days of age because of a
large tumour growth on the head. Dissection revealed tumour tissue in the
brain and in the left kidney. GPI analysis showed EC cell contributions
(10-20%) to all tissues analysed (liver, gut, brain, heart, lungs, kidney, spleen,
blood, muscle) including the tumours. A small amount of eye pigmentation
was also seen.
The high rate of successful development and low rate of chimaerism observed
104
J. ROSSANT AND M. W. MCBURNEY
Table 2. GPI analysis of chimaeras produced by injection ofP19 aggregates into
blastocysts: 9'5-day conceptuses
GPI phenotype
A
Embryo No.
Embryo
Yolk sac
1
©
©
3
©
©
3
©
©
3
©
3
3
3
3
3
No embryo
©
O
O
©
3
3
©
©
©
©
©
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
No embryo
20
No embryo
21
No embryo
o
o
o
o
©
o
©
©
©
©
©
o
Trophoblast
©
O
o
o
o
©
©
©
©
o
o
©
o
©
o
3
3
O
©
©
O
Morphology
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Small, retarded
Small, retarded
Misshapen head
Open neural tube
Open neural tube
Open neural tube
Embryo disorganized, disintegrating
Embryo disorganized, disintegrating
Embryo disorganized, disintegrating
Embryo disorganized, disintegrating
Embryo disorganized, disintegrating
No embryo, Y.S. only
No embryo, Y.S. only
No embryo, Y.S. only
in this last experiment reflects the variation between experiments often observed
with PI9 cells. In some experiments, nearly all embryos were chimaeric and
abnormal while in others, most embryos were normal and non-chimaeric.
The reasons for this variability were not clear but may be related to the fact
that the P19 cultures were not necessarily of clonal origin. For subsequent
experiments, a rigorously cloned population of cells was used.
Potential of a euploid male teratocarcinoma cell line
105
Table 3. GPI analysis of chimaeras produced by injection of P19 aggregates into
blastocysts: 12-5-day conceptuses
GPI phenotype
Embryo No.
Y.S.
1
O
©
2
3
4
5
6
7
8
9
10
11
Li
Gut
Lu
He
Br
Car
o O©©©
oO© © ©
o a 3O3 3
o 3 3 33 3
o © ©©©3
© 3
o 3 ©© ©3 ©O ©©
o © ©©©©
o ©O©© 3 ©
o
i
o
Eye
pigment
Morphology
+
Normal
+
Normal
+
Loose tissue on snout
+
Loose tissue on snout
+
Red marks on brain
+
Red marks on brain
—
Lump on head
+
Lump on head
+
Small lumps on head
Not analysed
+
Dead, neural tube open
Not analysed
+
Dead, brain open
Y.S., yolk sac; Li, liver; Lu, lungs; He, heart; UG, urogenital system; Br, brain;
Car, remainder of carcass.
Chimaeras produced by injection ofP19S18 aggregates into blastocysts
The results of these experiments were very similar to those obtained following
injection of P19 cells. The rate of chimaerism was very high in mid-gestation
(63%: Table 1). Most conceptuses were analysed at 9-5-10-5 days of development (Table 4) and EC cell contributions to embryonic tissues were often large
(up to 65 %). Again, many foetuses were grossly abnormal, with neural tube
defects predominating. Two chimaeras dissected at 12-5 days of development
showed EC cell contributions (up to 50%) to all tissues analysed and also
possessed eye pigmentation. The forebrain region was open and grossly
deformed in both.
All four live chimaeras detected at term were abnormal. Three mice, with
large tumour-like growths on the head and a little pigment in the eyes, died
and were eaten so that detailed GPI analysis could not be performed. The
fourth chimaera had no obvious pigment in the eyes, but a large tumour was
dissected from the brain region when the animal was killed at 3 weeks of age
GPI analysis revealed low EC cell contributions to the lungs and blood as well
as to the tumour. No other tissue was chimaeric.
106
J. ROSSANT AND M. W. MCBURNEY
Table 4. GPI analysis of chimaeras produced by injection of PI9S18 aggregates
into blastocysts: 9-5 to 10-5-day conceptuses
GPI phenotype
A
Embryo No.
Embryo
Yolk sac
Trophoblast
1
~»
©
3
©
3
O
©
©
©
O
O
O
O
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
3
3
©
©
3
3
©
3
3
3
(3
©
3
3
3
o
o
©
©
o
o
o
Not analysed
Not analysed
O
O
o
o
o
o
Not analysed
O
O
o
o
©
©
©
o
o
©
o
o
o
o
©
o
©
©
o
o
o
o
Morphology
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Small, retarded
Small, retarded
Small, retarded
Open neural tube
Open neural tube
Open neural tube
Open neural tube
Open neural tube
Open neural tube
Embryo disorganized, disintegrating
Embryo disorganized, disintegrating
Embryo disorganized, disintegrating
Chimaeras produced by injection of P19S18 single cells into blastocysts
The high rate of chimaerism achieved in mid-gestation with injections of
P19S18 aggregates suggested that it would be feasible to test the potential of
single P19S18 cells by blastocyst injection. A fairly high number of mid-term
chimaeras (15 % of live conceptuses) was indeed produced following injection of
single P19S18 cells (Table 1, Table 5). Colonization of all the internal tissues
analysed was rare but there was no obvious pattern to the sets of tissues colonized in different chimaeras. Contributions of up to 50% of the carcass were
Potential of a euploid male teratocarcinoma cell line
107
Table 5. GPI analysis of chimaeras produced by injection of PI 9S18 single cells
into blastocysts: 12-5 to 15-5-days
GPI phenotype
Embryo No. Y.S.
3
4
5
6
7
8
9
10
11
12
13
14*
15
16
17
18*
19
20
Li
Gut
Lu
He UG
Br
Car
Eye
pigment
—
—
—
+
+
+
—
Not analysed
Not analysed
+
+
+
—
+
+
+
+
+
+
+
+
Morphology
Normal
Normal
Normal
Normal
Red marks on head
Red marks on head
Red marks on head
Red marks on head
Loose tissue on snout
Loose tissue on snout
Loose tissue on snout
Lumps on head
Lumps on head
Lumps on head
Open abnormal brain
Open abnormal brain
Open abnormal brain
Open brain, tumour growth
Dead, exencephaly
Dead, exencephaly
* enough tumour tissue for histology and GPI
Y.S., yolk sac; Li, liver; Lu, lungs; He, heart; UG, urogenital system; Br brain;
Car, remainder of carcass.
detected. All but six chimaeras also showed some eye pigmentation. Various
abnormalities of development were also observed, again mostly in the head
region, ranging from loose skin on the snout, through open fore-brain development, to tumour-like growths on the brain. No obvious abnormalities were
observed in other tissues, even where these proved to have high levels of EC
cell contribution by GPI. Four chimaeras showed no obvious abnormalities
at this stage of development.
Only one chimaera was born alive. This mouse was killed when 7 days old
because of a massive tumour-like growth in the head region. Tumour growths
were also found in the chest and testis. Eye pigmentation and a little coat pigmentation in the head region was also observed. GPI analysis revealed EC cell
contributions to the tumours and normal tissue of the brain and to unspecified
tissues in the carcass, but not to any of the major internal organs analysed.
108
J. ROSSANT AND M. W. MCBURNEY
y
spm
X
Fig. 1. Section of brain tumour from mouse derived from blastocyst injected with
15-25 P19 cells. EC cells and striated muscle are present.
Fig. 2. Section of kidney tumour from the same mouse as Fig. 3. Most cells appear
to be EC-like. Degenerating kidney tubules (arrow) are present.
Fig. 3. Section of a brain tumour from mouse derived from blastocyst injected
with a single P19S18 cell. In this section, cartilage (arrow) is clearly differentiated
and other undefined cell types are also present.
Grid bar represents 50 fim.
Potential of a euploid male teratocarcinoma cell line
109
Tumour histology
It was often difficult to distinguish whether the abnormal morphology of
mid-gestation foetuses was caused by tumour growth or by disorganization of
normal development. However, two chimaeras from P19S18 single cell injections clearly had tumour-like growths on the brain. Histological examination
of these revealed a mass of undifferentiated, EC-like cells only. Tumours were
also recovered from three mice dissected after birth. Mouse 1, derived from a
blastocyst injected with an aggregate of PI9 cells, had a tumour on the brain
which apparently consisted of EC cells and various differentiated tissues
including striated muscle (Fig. 1). It also had a tumour in the kidney which
consisted entirely of EC-like cells, which were invading the kidney tissue (Fig.
2). Mouse 2, derived from a blastocyst injected with a P19S18 aggregate, had a
tumour on the brain which was clearly a teratocarcinoma, consisting of EC
cells and various differentiated tissues, including cartilage. Mouse 3, derived
from a blastocyst injected with a single P19S18 cell had a teratocarcinoma on
the brain (Fig. 3), and a tumour in the region of the thymus which seemed to
consist of EC cells only. This mouse also showed invasion of EC-like cells into
the tissues of the testis.
DISCUSSION
The karyotypically normal male EC cell line, PI9, and a clonal sub-line,
P19S18, readily colonized the host embryo following blastocyst injection and
produced large numbers of chimaeras in mid-gestation. The rates of chimaerism
were 50-60% for aggregates of P19 and P19S18 cells and 15% for single
P19S18 cells. Both rates approach those produced by injection of similar
numbers of ICM cells (Gardner, 1968; Gardner & Lyon, 1971; Papaioannou &
Gardner, 1979; Rossant & Lis, 1979). Levels of EC cell contribution to individual chimaeras were also high, up to 50-60% in some cases, including
some chimaeras produced by single cell injection. EC cell contributions to a
wide variety of normal tissues were observed. However most chimaeras also
showed various degrees of morphological abnormality. The degree of abnormality seemed to correlate roughly with the level of EC cell contribution (Tables
2-5). Some foetuses were completely disorganized, while others showed restricted areas of abnormal development, mostly in the brain and head region. Most
of these abnormalities would have been incompatible with survival to term and
only six live chimaeras were produced. All showed EC cell contributions to
some normal tissues, including, in most cases, the pigmented retinal epithelium,
but each one also contained tumour growths in the brain and elsewhere. Some
of these tumours were clearly teratocarcinomas, while all appeared to contain
undifferentiated, EC-like cells. Thus, P19 and P19S18 can produce chimaeras
in mid-gestation at a very high rate but the EC cells do not appear to behave
completely like normal embryonic cells. EC cells can contribute to normal
110
J. ROSSANT AND M. W. MCBURNEY
embryonic tissues and to abnormal tissue development and tumour formation
in the same mouse.
The design of the current experiments allowed us to eliminate some explanations for the incomplete regulation of P19 cells by the embryonic environment.
Variability in the original, uncloned culture of P19 cannot entirely explain the
colonization of both normal and abnormal tissues by P19 aggregates, since cell
aggregates from the cloned sub-line, P19S18, produced identical patterns of
colonization of host embryos. Incomplete regulation of in vitro EC cells has
been reported previously after injection of large numbers of EC cells into
blastocysts (Papaioannou et al. 1978; Papaioannou, 1980) and, based on these
and other observations, it has been suggested that there may be a limit to the
number of cells which can be regulated by a host blastocyst (Dewey et al. 1978;
Illmensee, 1978; Pierce et al. 1979; Martin, 1980). The rates of chimaerism
produced by previous in vitro cell lines have been too low to allow this hypothesis to be tested by following the fate of single EC cells after blastocyst
injection (Papaioannou, 1980). However, such experiments were possible with
P19S18, since it produced chimaeras at a very high rate. These experiments
showed that even single P19S18 cells were not completely regulated by the
host environment. A single P19S18 cell could contribute to both normal and
abnormal or tumorigenic tissue in the same mouse. Incomplete normalization
seems, therefore, to be an intrinsic property of this particular cell line, and may
relate to its poor response to in vitro conditions normally conducive to differentiation. Low concentrations of certain drugs are required to induce differentiation (Jones-Villeneuve et al. 1982; McBurney, Jones-Villeneuve, Edwards &
Anderson, in preparation), suggesting that the cells may respond inefficiently
to developmental signals and proliferate as EC stem cells in the embryonic
environment.
Thus, only one (METT-1, Stewart & Mintz, 1981) out of five euploid EC
cell lines tested so far has proved capable of responding normally to developmental signals and contributing to the germ line as well as all somatic tissues.
Even with METT-1 injections, one chimaera did contain a teratocarcinoma,
indicating that regulation may not always be complete. The potential of single
METT-1 cells has not yet been assessed. P19, although capable of colonizing
the embryo at a higher rate than most other cell lines, almost always showed
incomplete regulation. Thus, while euploidy of EC cells is presumably necessary
for normal differentiation of somatic and, particularly, germ cells, it is not a
sufficient prerequisite for such differentiation (Papaioannou et al. 1979).
Subtle genetic alterations not revealed by banding or epigenetic changes in
responses to gene regulation may occur during isolation of EC cell lines and
prevent regulated integration of EC cell progeny into the embryo.
We should like to thank Dr V. E. Papaioannou for useful discussion. This work was
supported by grants from the Natural Sciences and Engineering Research Council of Canada
(J. R.) and the Medical Research Council of Canada and the National Cancer Institute of
Canada (M.W.M.).
Potential of a euploid male teratocarcinoma cell line
111
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{Received 12 October 1981, revised 7 February 1982)