/. Embryol. exp. Morph. Vol. 38, pp. 63-75, 1977
Printed in Great Britain
63
Mouse teratomas and embryoid bodies: their
induction and differentiation
By S. A. ILES 1
From the Zoology Department, Oxford
SUMMARY
Teratomas were induced by the transfer of mouse blastocysts (C3H and 129/J strains) and
egg-cylinders (C3H) to extra-uterine sites. C3H and 129/J blastocysts cultured in vitro fox: A
or 5 days could also form teratomas in extra-uterine sites.
Four transplantable teratomas, or teratocarcinomas, were derived from C3H embryos;
embryoid bodies were derived from each line. The differentiative capacity of a teratocarcinoma was shown to be similar whether it was maintained as a solid tumour or as embryoid
bodies.
INTRODUCTION
Both ovarian and testicular teratomas occur spontaneously in certain mouse
strains (LT and 129/J respectively); they can also be induced by grafting to
extra-uterine sites embryos up to the 8th day of development (129/J, C3H,
C57BL, CBA, AKR and A/He strains), or male embryonic genital ridges (129/J
and A/He strains) (reviewed by Solter, Damjanov & Koprowski, 1975). A
proportion of spontaneous (129/J) and embryo-derived (129/J, C3H, A/He and
129/J x A/He Fx hybrid) teratomas have been reported to be transplantable for
a number of generations (Stevens, 1958, 1970; Damjanov, Solter, Belicza &
Skreb, 1971 b); these transplantable teratomas, or teratocarcinomas, contain
embryonal carcinoma cells (ECC) which are thought to be the stem cells for
growth at each transplant generation. When 129/J teratocarcinomas are converted to the ascites form, the peritoneal fluid contains multi-cellular bodies:
these bodies typically consist of a core of ECC, surrounded by endoderm, and
are called embryoid bodies (Stevens, 1959; Pierce & Dixon, 1959).
Teratocarcinomas arise only from grafted embryos not older than 8th day
egg-cylinders: older embryos will only form small benign teratomas (Damjanov,
Solter & Skreb, 1971 a). Attempts were therefore made to induce tumours from
blastocysts cultured for 4-6 days, so as to investigate (i) if it is the age or the
state of organization of the embryo which determines its ability to form a
benign teratoma or a teratocarcinoma, (ii) if a higher incidence of tumours can
be obtained from cultured blastocysts as opposed to untreated blastocysts or
egg-cylinders.
1
Author's address: Department of Zoology, South Parks Road, Oxford, 0X1 3PS, U.K.
5-2
64
S. A. ILES
Until now, embryoid bodies have only been reported in 129/J mice: this paper
describes the derivation of embryoid bodies from C3H teratocarcinomas. A
comparison has been made between the developmental capacity of teratocarcinomas maintained as solid tumours or as embryoid bodies. Such a comparison
provides information about the relationship between growth conditions and the
maintenance of pluripotentiality.
Cell lines have been derived from some of the C3H teratocarcinomas and
embryoid bodies described in this paper. Cells from some of the tumours are
capable of colonizing the mouse blastocysts and forming part of the animal
which is born (Papaioannou, McBurney, Gardner & Evans, 1975).
MATERIALS AND METHODS
(i) Induction of tumours
Blastocysts were flushed from the uterus and teased from the oviducts of C3H
and 129/J females on the 4th day of pregnancy (day of finding the copulation
plug= 1st day of pregnancy). Egg-cylinder stages (7th and 8th day of pregnancy)
were dissected from uterine decidual swellings and separated from the ectoplacental cone and primary trophoblast. Embryos were transferred beneath the
kidney or testis capsule of syngeneic adult recipients with a glass micropipette,
controlled by a mouth-piece via a length of flexible polythene tubing. Recipients
were anaesthetized with Avertin (Winthrop, U.S.A.) at 0-01 ml of 2-5 % Avertin
per gram body weight. Transfer sites were inspected 2-3 months after transfer,
unless stated otherwise.
(ii) Transplantation of tumours and induction of embryoid bodies
Solid tumours were chopped finely with scissors in sterile PBS (Dulbecco ' A '
from Dulbecco & Vogt, 1954). 0-5 ml of this tumour suspension was injected
subcutaneously or intraperitoneally with a trochar to syngeneic or semisyngeneic (129/J xC3H Fx) adult recipients under ether anaesthesia. After
intraperitoneal (IP) passage of a tumour for a few generations, ascites fluid was
sometimes found in the peritoneal cavity in addition to solid implants of the
tumour. This ascites fluid was found to contain embryoid bodies. The embryoid
bodies could be maintained by IP injection of ascites fluid to syngeneic or semisyngeneic recipients. Solid tumours could also be obtained by subcutaneous
(SC) injection of ascites fluid or embryoid bodies washed free of blood and
suspended in sterile PBS.
In the case of short-term growths of 129/J blastocysts in the kidney, the whole
growth was dissected out of the kidney and transferred to the kidney of another
recipient with a wide micropipette.
Induction and differentiation of mouse teratomas
65
(iii) Culture of embryos for transfer
(a) NCTC medium. Blastocysts were cultured overnight in V medium (Whitten,
1971) then transferred to drops of NCTC-109 medium (Evans, Bryant, Kerr
& Schilling, 1964; Biocult Laboratories, Paisley, Scotland) supplemented with
10 % foetal calf serum (FCS) (Chew & Sherman, 1975) under paraffin oil (Boots'
liquid paraffin, B.P., U.K.) in glass dishes.
(b) oc medium. Blastocysts were transferred directly to a medium (Stanners,
Eliceiri & Green, 1971) supplemented with 10% FCS either in drops under
paraffin oil in glass dishes, or in plastic dishes (Falcon Plastics or Sterilin) without oil. Some blastocysts were denuded of the zona pellucida with 0-5 % pronase
and incubated in 0-05 % trypsin in PBS for 30 min before culture (Pienkowski,
Solter & Koprowski, 1974).
In both (a) and (b), embryos were cultured for a total of 4-6 days in a humidified atmosphere of 5 % CO2 in air at 37 °C. Before transfer to extra-uterine sites,
they were detached from the culture vessel with a glass micropipette.
(iv) Histology
Growths were fixed in Bouin's fluid or formol saline, embedded in paraffin
wax (M.P. 56 °C) and sectioned at 8 ^m. Sections were stained in alcian blue
at pH 2-5 followed by Masson's trichrome (Evans, 1972). One in ten sections of
each tumour was scanned, but every section of the small growths was inspected.
RESULTS
(i) Induction of tumours
(a) 1291J. Ten per cent of 129/J blastocysts (3/28) gave rise to teratomas of at
least half the size of the host organ in the testes, but 129/J blastocysts failed to
develop into tumours of a similar size or tissue composition in the kidney.
Seven out of 34 blastocysts transferred to the kidney formed small nodules 1-2
mm in diameter. Five of these were fixed and sectioned: one contained an epithelial cyst lined by secretory epithelium, while the others contained yolk-sac
material, cells resembling trophoblast giant cells and other unidentifiable cells.
In order to investigate this failure of 129/J blastocysts to form teratomas in the
kidney, hosts were killed 6 days after blastocyst transfer, and the kidneys were
examined. In 16 out of 26 recipients, a haemorrhagic spot about 2 mm in
diameter was found at the site of transfer. Two of these growths were fixed
and sectioned: one consisted of well organized embryonic and extra-embryonic
structures, while the other consisted of trophoblast giant cells and extra-embryonic tissues. The failure of 129/J blastocysts to form teratomas in the kidney
cannot therefore be due to their inability to start to grow in this site.
Ten of these early kidney growths were retransferred to the kidneys of a second
series of hosts, on the assumption that the disturbance caused by the second
66
S. A. ILES
Table 1. Development of cultured 129\J and C3H blastocysts after
transfer to the testis
Strain
Medium
129/J
129/J
129/J
129/J
129/J
129/J
129/J
NCTC
C3H
C3H
C3H
C3H
C3H
C3H
C3H
a
a
a
a
a
a
NCTC
a
a
a
a
a
a
Pronase and
trypsin
—
—
—
Yes
Yes
Yes
—
—
—
—
Yes
Yes
Yes
Number of
Number
of
days in Number of
culture blastocysts teratomas
4-6
4
5
6
4
5
6
4-6
4
5
6
4
5
6
27
10
16
13
13
14
9
52
10
8
8
18
11
8
0
3
3
0
3
0
0
1
3
1
0
3
0
0
(%)
(0)
(30)
(19)
(0)
(23)
(0)
(0)
(2)
(30)
(12)
(0)
(17)
(0)
(0)
transfer might induce the disorganization of ordered embryonic structures that
precedes teratoma formation (Stevens, 1970). No tumours were found 2 months
after second transfer.
It was found that teratomas can sometimes develop from the outgrowths
arising from cultured blastocysts. Blastocysts cultured in NCTC and oc media
hatched from the zona pellucida and attached to the bottom of the culture dish:
trophoblast giant cells spread out as a monolayer, while the inner cell mass
(ICM) developed into a knob-like structure. This ICM 'knob' often developed
into a two-layered structure resembling an egg-cylinder (as found by Pienkowski
et ah (1974) and Spindle & Pedersen (1973). Outgrowth of blastocysts in a
medium was superior to that in NCTC medium. Only embryos with good ICM
development were selected for transfer to the testis, i.e. good proliferation of the
ICM, but not always with development of two distinct layers.
A proportion of the blastocysts cultured in a medium gave rise to teratomas
after transfer to the testis, but none of the blastocysts cultured in NCTC medium
did so (Table 1).
(b) C3H. Thirty-six per cent of C3H blastocysts (9/25) formed teratomas in
the testes of adult hosts. Egg-cylinder stages of C3H embryos also gave rise to
teratomas in the testis: 33 % of the 7th day embryos (9/26) and 66 % of 8th day
embryos (25/38) formed teratomas. One out of three 7th day embryos transferred
to the kidney formed a teratoma.
Outgrowth of C3H blastocysts in culture was similar to that of 129/J blastocysts, with superior outgrowth in a medium, but outgrowths were not as large
as those obtained from 129/J blastocysts. A number of blastocysts cultured in
Induction and differentiation of mouse teratomas
67
Table 2. Histology of primary tumours
C3H 7th
Origin of tumour
No. of tumours
129/J be*
...
3
ECC
lOOf
Nervous tissue
Pigment
Keratin, epithelium
Glandular epithelium
Ciliated epithelium
Simple epithelium
Cartilage
Bone
Smooth muscle
Striated muscle
Adipose tissue
Yolk-sac
carcinoma
66
33
100
66
100
100
33
100
100
100
66
0
129/J
cult, be
9
33$
100
55
100
100
100
89
33
33
55
78
44
0
C3H 8th
day
day
C3H
C3Hbc
embryo
embryo
cult, be
8
75
75
37
87
87
75
75
25
12
62
75
37
12
10
60
90
90
90
90
80
100
80
80
80
90
40
0
24
62
96
75
87
75
71
75
54
54
79
79
33
0
8
75$
100
62
87
75
87
87
62
62
62
87
25
0
* bc= blastocyst.
t Figures in the columns refer to the percentage of tumours showing a particular tissue.
% ECC were present mainly in small numbers, and their identification was often uncertain.
a medium gave rise to teratomas after transfer to the testis, whereas only one of
the blastocysts cultured in NCTC medium did so (Table 1).
(ii) Histology of primary tumours
Table 2 shows the distribution of tissues in the primary teratomas referred
to in Section (i) of the Results. Since only part of each tumour was available for
histology (1/20 to 1/100 of each tumour) the absence of a particular tissue from
the sections examined does not mean that it could not have been present in
some other part of the tumour. In a well differentiated tumour, however, most
tissues could be found in the first few sections examined, so the absence of a
tissue from the available sections makes it very likely that it is absent from the
rest of the tumour, or only present in very small amounts.
A survey of histology of these primary tumours shows that tumours derived
from 129/J blastocysts were not grossly different from those derived from C3H
blastocysts. Some differences could be seen in tumours derived from C3H
blastocysts and those derived from C3H egg-cylinder stages: although a full
range of differentiated tissues could be found in both types of tumour, more
egg-cylinder-derived tumours showed the full range of tissues than did blastocyst-derived tumours (this was particularly noticeable for pigment, cartilage
and bone), while yolk-sac carcinoma was found only in a tumour arising from
a blastocyst (but see Section (iv) for appearance of yolk-sac carcinoma in
transplants of an egg-cylinder-derived tumour).
08
S. A. ILES
Table 3. Tissues produced by transplant generations o/ tumour
Generation
...
P
1
2
3*
4
5
6
7
8
9
10
No. of tumours ...
1
ECC
It
Nervous tissue
Pigment
Keratin, epithelium
Glandular epithelium
Ciliated epithelium
Simple epithelium
Cartilage
Bone
Smooth muscle
Striated muscle
Adipose tissue
Yolk-sac carcinoma
1
1
1
1
1
1
1
1
1
1
1
0
2
2
2
1
1
2
2
2
1
2
2
1
0
0
4
4
4
2
4
4
3
4
3
4
1
4
0
0
4
3
3
0
4
4
2
2
3
2
3
2
0
0
3
3
2
0
2
2
3
3
1
0
1
1
0
2
5
5
5
0
3
2
2
3
1
2
1
2
1
1
5
5
3
0
3
2
3
3
3
3
2
3
2
4
1
1
0
0
0
0
0
1
1
1
0
0
1
1
3
3
1
0
2
1
1
3
2
2
2
1
2
3
1
1
0
0
0
0
0
1
1
0
0
0
0
0
2
2
1
2
2
1
1
2
2
2
1
0
0
1
P = primary tumour.
* Embryoid bodies first appeared in this generation.
f Figures in columns refer to number of tumours forming a particular tissue.
Tumours from cultured blastocysts showed a range of tissues similar to that
found in tumours from untreated blastocysts or egg-cylinders, but ECC, where
present, were found only in very small numbers and were hard to identify with
certainty. Tumours from C3H-cultured blastocysts resembled tumours from C3H
egg-cylinders more strongly than they did tumours from C3H blastocysts in
terms of the extent to which they were differentiated.
(iii) Transplantability of tumours
None of the three primary tumours derived from 129/J blastocysts was
transplantable, but transplantable tumours were obtained from C3H blastocysts and egg-cylinders. Not all tumours that grew at the 1st transplant generation continued to grow in subsequent generations, but permanent lines have
been established from all tumours that continued to grow after the 1st transplant. The numbers of C3H tumours still growing are as follows: one out of
five tumours from blastocysts transplanted, two out of five tumours from 7th
day embryos and one out of 17 tumours from 8th day embryos.
(iv) Progression of transplantable tumours
The histology of the four C3H tumours maintained as transplantable teratocarcinomas has been examined during a number of transplant generations so as
to discover whether these would become restricted in their capacity to differentiate. Age of tumours is dated from the time of writing (September 1975).
Tumour 17 is derived from a C3H 7th day embryo transferred to the testis
2\ years ago; the primary tumour consisted of a wide range of differentiated
Induction and differentiation of mouse teratomas
69
Table 4. Tissues produced by transplant generations of tumour 86
Generation
...
No. of tumours
...
ECC
Nervous tissue
Pigment
Keratin, epithelium
Glandular epithelium
Ciliated epithelium
Simple epithelium
Cartilage
Bone
Smooth muscle
Striated muscle
Adipose tissue
Yolk-sac carcinoma
P
1
2*
3
4
5
6
1
2
2
2
1
2
2
2
2
2
2
2
2
2
0
2
2
2
G
0
0
0
0
1
1
0
1
0
0
2
2
2
0
0
0
1
1
0
0
0
0
0
0
2
2
2
0
1
1
1
0
0
0
0
0
1
0
4
4
3
0
0
0
0
3
0
0
0
0
1
0
3
3
3
1
2
3
2
0
0
0
0
0
0
0
It
1
1
1
1
1
1
1
1
1
1
0
0
P = primary tumour.
* Embryoid bodies first appeared in this generation.
t Figures in column refer to number of tumours forming a particular tissue.
tissues as well as ECC. At the 10th generation, it was still producing all the
tissues found in the primary tumour except striated muscle, with the addition of
yolk-sac carcinoma (see Table 3). Yolk-sac carcinoma first appeared in two
sub-lines in the 4th generation: it is known that yolk-sac carcinoma can arise
from embryonal carcinoma cells as well as from embryonic cells from the eggcylinder (Damjanov & Solter, 1973). In the 5th and 6th generations, some sublines produced only ECC and nervous tissue, but only the sub-lines producing
ECC and a wide range of differentiated tissues were transplanted further. All
the later generations of tumour 17 were dominated by ECC.
Tumour 86 grew from a C3H 8th day embryo, and has now been maintained
for If years. The primary tumour contained a wide range of differentiated
tissues together with ECC. During the 2nd transplant generation, a progressive
simplification of the tissues produced occurred. In subsequent generations,
tumours consisted mainly of ECC and nervous tissue, together with small areas
of epithelial and adipose tissue (see Table 4).
Tumour 106, derived from a C3H blastocyst transferred to the testis If years
ago, progressed in a manner similar to that of tumour 86. Fewer differentiated
tissues were produced in the 2nd and 3rd generations and by the 4th generation
ECC and nervous tissue predominated, with small areas of epithelium in some
tumours (see Table 5).
Tumour 145 was derived from a C3H 7th day embryo transferred to the kidney
only 8 months ago. The primary tumour contained ECC and a wide range of
differentiated tissues. It remained well differentiated in the 2nd and 3rd generations but two sub-lines appeared in the 4th generation: one consisted only of
s. A.
7U
ILES
Table 5. Tissues produced by transplant generations of tumour 106
Generation ...
No. of tumours
...
EEC
Nervous tissue
Pigment
Keratin, epithelium
Glandular epithelium
Ciliated epithelium
Simple epithelium
Cartilage
Bone
Smooth muscle
Striated muscle
Adipose tissue
Yolk-sac carcinoma
P
1
2*
3
4
5
6
7
1
1
1
1
1
1
1
1
1
0
1
1
1
0
0
2
2
1
0
0
2
0
0
0
0
1
0
0
0
2
2
2
0
0
1
0
1
1
0
2
1
0
0
5
5
5
0
1
0
0
1
0
0
0
0
0
0
4
4
4
0
0
1
0
0
0
0
0
0
0
0
4
4
4
0
0
0
0
1
0
0
0
0
0
0
1
1
1
0
0
1
0
1
0
0
0
It
1
0
1
1
1
1
1
1
1
1
1
0
n
1
0
P = primary tumour.
* Embryoid bodies first appeared at this generation.
f Figures in the columns refer to the number of tumours forming a particular tissue.
% This unexpected appearance of striated muscle in the 7th generation may have been due
to the inclusion of some body wall muscle with the tumour when it was cut out, although
this muscle did not have the usual appearance of included body wall muscle.
ECC and nervous tissue with a little ciliated epithelium, while the other
remained well differentiated.
(v) Derivation of embryoid b odies from C3H teratocarcinomas
Intraperitoneal transfer of all four lines of C3H teratocarcinomas always
resulted in the formation of ascites fluid containing structures resembling the
embryoid bodies arising from 129/J teratocarcinomas (Stevens, 1959, 1970). In
tumours 86 and 106, embryoid bodies appeared after IP transfer of 1st transplant generation solid tumour, while in tumours 17 and 145, embryoid bodies
appeared one generation later.
These C3H embryoid bodies could be maintained by intraperitoneal passage
of ascites fluid (see Methods section). Ascites fluid with embryoid bodies also
regularly appeared after IP transfer of later generations of solid tumour. Conversely, implants of solid tumour were often found after transfer of ascites fluid
containing embryoid bodies.
Transplantability of C3H embryoid bodies appeared to increase with subsequent generations. For example, the earliest C3H 17 ascitic fluid was transplanted to 12 recipients: four developed ascites fluid with embryoid bodies,
seven failed to develop ascites fluid and one died. Later generations of embryoid
bodies were consistently transplantable.
The structure of the embryoid bodies of lines 17, 86 and 106 was studied soon
after their derivation and after at least one year. The early embryoid bodies of
Induction and differentiation of mouse teratomas
A
,
,
MJT
B
Fig. 1. Embryoid bodies dervied from C3H teratocarcinomas.
(A) Embryoid body derived from tumour 17.
(B) 'EndodermaP vesicle derived from tumour 17.
(C) Early embryoid body from tumour 106.
(D) Embryoid body from tumour 86.
(E) Embryoid body from tumour 145. Scale bar = 50/*m.
71
72
S. A. ILES
tumour 17 consisted of masses of ECC and lacked a distinct outer layer; they
were round or oval in cross-section. One year later, their structure was very
similar (Fig. 1 A). Solid tumour implants found in the peritoneal cavity after IP
transfer of these embryoid bodies give some indication of the potency of these
embryoid bodies: some of these implants from C3H 17 embryoid bodies contained ECC and a range of differentiated tissues, others contained ECC, yolk-sac
carcinoma, nervous and epithelial tissues, while some consisted only of ECC.
More recently, the embryoid bodies of tumour 17 have undergone a distinct
change in morphology: they are now small hollow vesicles of flattened cells
resembling parietal yolk-sac, either empty or filled with material with the
staining characteristics of Reichert's membrane material (Fig. 1B). Injection of
these embryoid bodies to the subcutaneous space gives tumours consisting
entirely of yolk-sac carcinoma.
The early embryoid bodies derived from tumours 86 and 106 were similar in
structure to early embryoid bodies from tumour 17, and their structure has not
so far changed with time. However, some early embryoid bodies of tumour 106
appeared to have a more distinct outer layer of flattened cells (Fig. 1C), resembling endoderm. This outer layer was very distinct in some later embryoid bodies
of tumour 86, with some of the inner cells organized into stuctures resembling
early ectoderm (Fig. 1D). Solid tumours arising after subcutaneous injection of
both 86 and 106 embryoid bodies containing only ECC and nervous tissue.
Embryoid bodies arising from tumour 145 consisted of masses of ECC, often
surrounded by a layer of flattened cells (Fig. 1E). Subcutaneous injection of
these embryoid bodies gave tumours consisting mainly of ECC, together with
neuro-ectodermal tubules and simple cuboidal epithelium; these tumours
resembled the subline of tumour 145 in which differentiative capacity was
restricted.
DISCUSSION
1. Induction of teratomas
C3H embryos form teratomas more frequently as they are transferred to the
testis at progressively later stages of development up to the 8th day. The proportions forming teratomas are: no one-cell eggs (lies et al. 1975), one-third of blastocysts and 7th day egg-cylinders, and two-thirds of 8th day egg-cylinders. Several
factors could account for the poorer growth of embryos earlier than the 8th day.
Their low cell number could reduce their chances of growing, and their development may be impeded by haemorrhages resulting from trophoblast growth;
more of the trophoblast precursors in the extra-embryonic ectoderm (Gardner
& Papaioannou, 1975) are likely to be removed during the dissection of the ectoplacental cone from 8th day embryos, and these embryos grow more frequently.
It is known that the majority of blastocysts transferred to extra-uterine sites
start to grow (e.g. Kirby, 1963, and 129/J transfers to the kidney in this study).
However, the incidence of teratoma formation from blastocysts shows that only
Induction and differentiation of mouse teratomas
73
few of these growths develop into teratomas. The culture of blastocysts
increases cell number and disorganizes tissue relationships as the trophoblast
layer spreads out on the substrate: it was therefore hoped that a higher frequency of teratoma formation would be obtained from cultured blastocysts.
Such an increase would be of value in the induction of teratomas from parthenogenetic blastocysts (lies et al. 1975) which rarely go on to form egg-cylinders in
vivo. Culture for 4 days in a medium did not raise the incidence of teratoma formation by C3H blastocysts above the original one-third, but it increased the
incidence with 129/J blastocysts from 10 % (Stevens, 1970, and this study) to
30 %; enzymic pre-treatment lowered the incidence of tumour formation from
both C3H and 129/J blastocysts cultured for 4 days in a medium, while longer
periods of culture greatly reduced tumour formation in both strains. This effect
of time is not understood, because embryos of the same total age can form
benign teratomas (Damjanov et al. 1971a). Tumours derived from blastocysts
cultured for 4 or 5 days do not contain significant amounts of ECC, but they do
show a wide range of differentiated tissues.
2. Development of teratocarcinomas and embryoid bodies
In this study, teratocarcinomas (transplantable teratomas) were obtained from
C3H blastocysts, 7th and 8th day egg-cylinders; similar tumours have been
obtained from C3H 8th day egg-cylinders by Damjanov et al. (1971 b) and from
129/J blastocysts and 7th day embryos by Stevens (1970). Both spontaneous
and embryo-derived teratocarcinomas may either retain the ability to differentiate into many tissues (pluripotent) or their differentiation may be restricted to
one or a few tissues (e.g. Stevens, 1958, 1970; Damjanov et al. 19716). The
histology of these C3H teratocarcinomas on successive transplant generations
shows that 17 retains pluripotency while 86,106 and 145 have become restricted.
The restriction of developmental potential of some tumours may be due to
the selection of rapidly dividing ECC whose capacity to differentiate has become restricted: such restrictions may be associated with abnormal karyotypes
(see following paper by lies & Evans, 1977). It is important to discover whether
teratocarcinomas maintained as embryoid bodies are more or less restricted
than those maintained as solid tumours. This paper is the first published description of embryoid bodies derived from C3H teratocarcinomas; their structure is
comparable with the simpler of the embryoid bodies in OTT 6050 ascites fluid
described by Stevens (1970).
It has been shown here that the developmental capacity cf a teratocarcinoma
is generally similar whether it is maintained as embryoid bodies or as a solid
tumour. Embryoid bodies of 86,106 and 145 all gave subcutaneous tumours
consisting of embryonal carcinoma and nervous tissue; these tumours strongly
resembled the final state of the solid tumour transplants. For 86 and 106, this
restriction in developmental capacity could be related to the observation that
solid tumours arising in the generation in which the embryoid bodies first
74
S. A. ILES
appeared were, in both cases, starting to show a similar restriction in their
capacity to differentiate (see Results); solid tumours of 145 became restricted (in
one subline) one generation later than the embryoid bodies first appeared.
Tumour 17 continued to differentiate well, and some solid implants found on
the wall of the peritoneum after injection of embryoid bodies developed a wide
range of differentiated tissues. However, the embryoid bodies of 17 have recently
become converted from the type of embryoid bodies found in 129/J mice
(Stevens, 1959,1970) and formed in vitro from teratocarcinoma cell lines (Martin
& Evans, 1975) to sacs of endoderm-like cells, which give rise to yolk-sac carcinomas when injected subcutaneously. A similar progression to yolk-sac carcinoma has been reported with strain 129/J embryoid bodies (Stevens, 1959;
Pierce & Dixon, 1959).
The conclusion from these observations is that tumours may become restricted
in developmental capacity whether they are maintained as solid tumours or as
embryoid bodies. Both growth conditions seem to select for cells with rapid
growth which may have restricted developmental capacities.
I should thank the following people: Dr C. F. Graham, for his advice and constant encouragement, Dr D. Solter, for reading the manuscript, and S. R. Bramwell, for expert
technical assistance. This work was supported by the Cancer Research Campaign and partly
by a Mary Goodger Scholarship.
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(Received 22 April 1976, revised 21 October 1976)