-
/ . Embryol. exp. Morph. Vol. 38, pp. 63-75, 1977 6 3
Printed in Great Britain
Mouse teratomas and embryoid bodies: theirinduction and
differentiation
By S. A. ILES1
From the Zoology Department, Oxford
SUMMARY
Teratomas were induced by the transfer of mouse blastocysts (C3H
and 129/J strains) andegg-cylinders (C3H) to extra-uterine sites.
C3H and 129/J blastocysts cultured in vitro fox: Aor 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 teratocarci-noma was shown to be
similar whether it was maintained as a solid tumour or as
embryoidbodies.
INTRODUCTION
Both ovarian and testicular teratomas occur spontaneously in
certain mousestrains (LT and 129/J respectively); they can also be
induced by grafting toextra-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/Jand A/He strains) (reviewed by
Solter, Damjanov & Koprowski, 1975). Aproportion of spontaneous
(129/J) and embryo-derived (129/J, C3H, A/He and129/J x A/He Fx
hybrid) teratomas have been reported to be transplantable fora
number of generations (Stevens, 1958, 1970; Damjanov, Solter,
Belicza &Skreb, 1971 b); these transplantable teratomas, or
teratocarcinomas, containembryonal carcinoma cells (ECC) which are
thought to be the stem cells forgrowth at each transplant
generation. When 129/J teratocarcinomas are con-verted to the
ascites form, the peritoneal fluid contains multi-cellular
bodies:these bodies typically consist of a core of ECC, surrounded
by endoderm, andare called embryoid bodies (Stevens, 1959; Pierce
& Dixon, 1959).
Teratocarcinomas arise only from grafted embryos not older than
8th dayegg-cylinders: older embryos will only form small benign
teratomas (Damjanov,Solter & Skreb, 1971 a). Attempts were
therefore made to induce tumours fromblastocysts cultured for 4-6
days, so as to investigate (i) if it is the age or thestate of
organization of the embryo which determines its ability to form
abenign teratoma or a teratocarcinoma, (ii) if a higher incidence
of tumours canbe obtained from cultured blastocysts as opposed to
untreated blastocysts oregg-cylinders.
1 Author's address: Department of Zoology, South Parks Road,
Oxford, 0X1 3PS, U.K.5-2
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64 S. A. ILES
Until now, embryoid bodies have only been reported in 129/J
mice: this paperdescribes the derivation of embryoid bodies from
C3H teratocarcinomas. Acomparison has been made between the
developmental capacity of teratocarci-nomas maintained as solid
tumours or as embryoid bodies. Such a comparisonprovides
information about the relationship between growth conditions and
themaintenance of pluripotentiality.
Cell lines have been derived from some of the C3H
teratocarcinomas andembryoid bodies described in this paper. Cells
from some of the tumours arecapable of colonizing the mouse
blastocysts and forming part of the animalwhich 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 C3Hand 129/J females on the 4th day of pregnancy (day
of finding the copulationplug= 1st day of pregnancy). Egg-cylinder
stages (7th and 8th day of pregnancy)were dissected from uterine
decidual swellings and separated from the ecto-placental cone and
primary trophoblast. Embryos were transferred beneath thekidney or
testis capsule of syngeneic adult recipients with a glass
micropipette,controlled by a mouth-piece via a length of flexible
polythene tubing. Recipientswere anaesthetized with Avertin
(Winthrop, U.S.A.) at 0-01 ml of 2-5 % Avertinper 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 injectedsubcutaneously or intraperitoneally
with a trochar to syngeneic or semi-syngeneic (129/J xC3H Fx) adult
recipients under ether anaesthesia. Afterintraperitoneal (IP)
passage of a tumour for a few generations, ascites fluid
wassometimes found in the peritoneal cavity in addition to solid
implants of thetumour. This ascites fluid was found to contain
embryoid bodies. The embryoidbodies could be maintained by IP
injection of ascites fluid to syngeneic or semi-syngeneic
recipients. Solid tumours could also be obtained by
subcutaneous(SC) injection of ascites fluid or embryoid bodies
washed free of blood andsuspended in sterile PBS.
In the case of short-term growths of 129/J blastocysts in the
kidney, the wholegrowth was dissected out of the kidney and
transferred to the kidney of anotherrecipient 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 with10 % 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 underparaffin oil in glass dishes, or in plastic
dishes (Falcon Plastics or Sterilin) with-out oil. Some blastocysts
were denuded of the zona pellucida with 0-5 % pronaseand 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 humidi-fied 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 paraffinwax (M.P. 56 °C) and sectioned at 8 ^m. Sections were
stained in alcian blueat pH 2-5 followed by Masson's trichrome
(Evans, 1972). One in ten sections ofeach 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 atleast half the size of the host organ in the testes,
but 129/J blastocysts failed todevelop into tumours of a similar
size or tissue composition in the kidney.Seven out of 34
blastocysts transferred to the kidney formed small nodules 1-2mm in
diameter. Five of these were fixed and sectioned: one contained an
epi-thelial cyst lined by secretory epithelium, while the others
contained yolk-sacmaterial, cells resembling trophoblast giant
cells and other unidentifiable cells.
In order to investigate this failure of 129/J blastocysts to
form teratomas in thekidney, hosts were killed 6 days after
blastocyst transfer, and the kidneys wereexamined. In 16 out of 26
recipients, a haemorrhagic spot about 2 mm indiameter was found at
the site of transfer. Two of these growths were fixedand sectioned:
one consisted of well organized embryonic and
extra-embryonicstructures, while the other consisted of trophoblast
giant cells and extra-embry-onic tissues. The failure of 129/J
blastocysts to form teratomas in the kidneycannot 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 secondseries of hosts, on the assumption that the
disturbance caused by the second
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66 S. A. ILES
Table 1. Development of cultured 129\J and C3H blastocysts
aftertransfer to the testis
Strain
129/J129/J129/J129/J129/J129/J129/JC3HC3HC3HC3HC3HC3HC3H
Medium
NCTCaaaaaa
NCTCaaaaaa
Pronase andtrypsin
———YesYesYes————YesYesYes
Number ofdays inculture
4-6456456
4-6456456
Number ofblastocysts
2710161313149
521088
18118
Numberof
teratomas
0330300
1310300
(%)
(0)(30)(19)(0)(23)(0)(0)
(2)(30)(12)(0)(17)(0)(0)
transfer might induce the disorganization of ordered embryonic
structures thatprecedes teratoma formation (Stevens, 1970). No
tumours were found 2 monthsafter second transfer.
It was found that teratomas can sometimes develop from the
outgrowthsarising from cultured blastocysts. Blastocysts cultured
in NCTC and oc mediahatched 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 developedinto a
two-layered structure resembling an egg-cylinder (as found by
Pienkowskiet ah (1974) and Spindle & Pedersen (1973). Outgrowth
of blastocysts in amedium was superior to that in NCTC medium. Only
embryos with good ICMdevelopment were selected for transfer to the
testis, i.e. good proliferation of theICM, but not always with
development of two distinct layers.
A proportion of the blastocysts cultured in a medium gave rise
to teratomasafter transfer to the testis, but none of the
blastocysts cultured in NCTC mediumdid so (Table 1).
(b) C3H. Thirty-six per cent of C3H blastocysts (9/25) formed
teratomas inthe testes of adult hosts. Egg-cylinder stages of C3H
embryos also gave rise toteratomas in the testis: 33 % of the 7th
day embryos (9/26) and 66 % of 8th dayembryos (25/38) formed
teratomas. One out of three 7th day embryos transferredto the
kidney formed a teratoma.
Outgrowth of C3H blastocysts in culture was similar to that of
129/J blasto-cysts, with superior outgrowth in a medium, but
outgrowths were not as largeas those obtained from 129/J
blastocysts. A number of blastocysts cultured in
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Induction and differentiation of mouse teratomas 67
Table 2. Histology of primary tumours
Origin of tumour
No. of tumours ...ECCNervous tissuePigmentKeratin,
epitheliumGlandular epitheliumCiliated epitheliumSimple
epitheliumCartilageBoneSmooth muscleStriated muscleAdipose
tissueYolk-saccarcinoma
129/J be*
3
lOOf6633
10066
10010033
100100100660
129/Jcult, be
9
33$10055
100100100893333557844
0
C3Hbc
8
75753787877575251262753712
C3H 7thday
embryo
10
609090909080
1008080809040
0
C3H 8thday
embryo
24
6296758775717554547979330
C3Hcult, be
8
75$10062877587876262628725
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 ofthe 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 referredto in Section (i) of the Results. Since only part
of each tumour was available forhistology (1/20 to 1/100 of each
tumour) the absence of a particular tissue fromthe sections
examined does not mean that it could not have been present insome
other part of the tumour. In a well differentiated tumour, however,
mosttissues could be found in the first few sections examined, so
the absence of atissue from the available sections makes it very
likely that it is absent from therest of the tumour, or only
present in very small amounts.
A survey of histology of these primary tumours shows that
tumours derivedfrom 129/J blastocysts were not grossly different
from those derived from C3Hblastocysts. Some differences could be
seen in tumours derived from C3Hblastocysts and those derived from
C3H egg-cylinder stages: although a fullrange of differentiated
tissues could be found in both types of tumour,
moreegg-cylinder-derived tumours showed the full range of tissues
than did blasto-cyst-derived tumours (this was particularly
noticeable for pigment, cartilageand bone), while yolk-sac
carcinoma was found only in a tumour arising froma blastocyst (but
see Section (iv) for appearance of yolk-sac carcinoma intransplants
of an egg-cylinder-derived tumour).
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0 8 S. A. ILES
Table 3. Tissues produced by transplant generations
Generation ...
No. of tumours ...ECCNervous tissuePigmentKeratin,
epitheliumGlandular epitheliumCiliated epitheliumSimple
epitheliumCartilageBoneSmooth muscleStriated muscleAdipose
tissueYolk-sac carcinoma
P
1
It111111111110
1
2
2211222122100
2
4
4424434341400
3*
4
3304422323200
4
3
3202233101102
5
5
5503223121211
6
5
5303233332324
o/ tumour
7
1
1000001110011
8
3
3102113222123
9
1
1000001100000
10
2
2122112221001
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 thatfound in tumours from untreated blastocysts or
egg-cylinders, but ECC, wherepresent, were found only in very small
numbers and were hard to identify withcertainty. Tumours from
C3H-cultured blastocysts resembled tumours from C3Hegg-cylinders
more strongly than they did tumours from C3H blastocysts interms of
the extent to which they were differentiated.
(iii) Transplantability of tumours
None of the three primary tumours derived from 129/J blastocysts
wastransplantable, but transplantable tumours were obtained from
C3H blasto-cysts and egg-cylinders. Not all tumours that grew at
the 1st transplant genera-tion continued to grow in subsequent
generations, but permanent lines havebeen established from all
tumours that continued to grow after the 1st trans-plant. The
numbers of C3H tumours still growing are as follows: one out offive
tumours from blastocysts transplanted, two out of five tumours from
7thday 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 terato-carcinomas has been examined during a number
of transplant generations so asto discover whether these would
become restricted in their capacity to differen-tiate. 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 testis2\ years ago; the primary tumour consisted of a wide
range of differentiated
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Induction and differentiation of mouse teratomas 69
Table 4. Tissues produced by transplant generations of tumour
86
Generation ...
No. of tumours ...ECCNervous tissuePigmentKeratin,
epitheliumGlandular epitheliumCiliated epitheliumSimple
epitheliumCartilageBoneSmooth muscleStriated muscleAdipose
tissueYolk-sac carcinoma
P
1
It111111111100
1
2
2212222222220
2*
222G0000110100
3
2
2200011000000
4
2
2201110000010
5
4
4300003000010
6
3
3312320000000
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 thetissues found in the primary tumour except
striated muscle, with the addition ofyolk-sac carcinoma (see Table
3). Yolk-sac carcinoma first appeared in twosub-lines in the 4th
generation: it is known that yolk-sac carcinoma can arisefrom
embryonal carcinoma cells as well as from embryonic cells from the
egg-cylinder (Damjanov & Solter, 1973). In the 5th and 6th
generations, some sub-lines produced only ECC and nervous tissue,
but only the sub-lines producingECC and a wide range of
differentiated tissues were transplanted further. Allthe later
generations of tumour 17 were dominated by ECC.
Tumour 86 grew from a C3H 8th day embryo, and has now been
maintainedfor If years. The primary tumour contained a wide range
of differentiatedtissues together with ECC. During the 2nd
transplant generation, a progressivesimplification of the tissues
produced occurred. In subsequent generations,tumours consisted
mainly of ECC and nervous tissue, together with small areasof
epithelial and adipose tissue (see Table 4).
Tumour 106, derived from a C3H blastocyst transferred to the
testis If yearsago, progressed in a manner similar to that of
tumour 86. Fewer differentiatedtissues were produced in the 2nd and
3rd generations and by the 4th generationECC and nervous tissue
predominated, with small areas of epithelium in sometumours (see
Table 5).
Tumour 145 was derived from a C3H 7th day embryo transferred to
the kidneyonly 8 months ago. The primary tumour contained ECC and a
wide range ofdifferentiated tissues. It remained well
differentiated in the 2nd and 3rd genera-tions but two sub-lines
appeared in the 4th generation: one consisted only of
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7U s
Table 5. Tissues produced by
Generation ...
No. of tumours ...EECNervous tissuePigmentKeratin,
epitheliumGlandular epitheliumCiliated epitheliumSimple
epitheliumCartilageBoneSmooth muscleStriated muscleAdipose
tissueYolk-sac carcinoma
P
1
It101111111110
1
1
1111111011100
. A. ILES
transplant
2*
2
2100200001000
generations
3
2
2200101102100
4
5
5501001000000
of tumour
5
4
4400100000000
106
6
4
4400001000000
7
1
1100101000
n10
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, althoughthis muscle did not have the usual
appearance of included body wall muscle.
ECC and nervous tissue with a little ciliated epithelium, while
the otherremained well differentiated.
(v) Derivation of embryoid b odies from C3H teratocarcinomas
Intraperitoneal transfer of all four lines of C3H
teratocarcinomas alwaysresulted in the formation of ascites fluid
containing structures resembling theembryoid bodies arising from
129/J teratocarcinomas (Stevens, 1959, 1970). Intumours 86 and 106,
embryoid bodies appeared after IP transfer of 1st trans-plant
generation solid tumour, while in tumours 17 and 145, embryoid
bodiesappeared one generation later.
These C3H embryoid bodies could be maintained by intraperitoneal
passageof ascites fluid (see Methods section). Ascites fluid with
embryoid bodies alsoregularly appeared after IP transfer of later
generations of solid tumour. Con-versely, implants of solid tumour
were often found after transfer of ascites fluidcontaining embryoid
bodies.
Transplantability of C3H embryoid bodies appeared to increase
with subse-quent generations. For example, the earliest C3H 17
ascitic fluid was trans-planted to 12 recipients: four developed
ascites fluid with embryoid bodies,seven failed to develop ascites
fluid and one died. Later generations of embryoidbodies were
consistently transplantable.
The structure of the embryoid bodies of lines 17, 86 and 106 was
studied soonafter their derivation and after at least one year. The
early embryoid bodies of
-
Induction and differentiation of mouse teratomas 71
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.
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72 S. A. ILES
tumour 17 consisted of masses of ECC and lacked a distinct outer
layer; theywere round or oval in cross-section. One year later,
their structure was verysimilar (Fig. 1 A). Solid tumour implants
found in the peritoneal cavity after IPtransfer of these embryoid
bodies give some indication of the potency of theseembryoid bodies:
some of these implants from C3H 17 embryoid bodies con-tained ECC
and a range of differentiated tissues, others contained ECC,
yolk-saccarcinoma, nervous and epithelial tissues, while some
consisted only of ECC.More recently, the embryoid bodies of tumour
17 have undergone a distinctchange in morphology: they are now
small hollow vesicles of flattened cellsresembling parietal
yolk-sac, either empty or filled with material with thestaining
characteristics of Reichert's membrane material (Fig. 1B).
Injection ofthese embryoid bodies to the subcutaneous space gives
tumours consistingentirely of yolk-sac carcinoma.
The early embryoid bodies derived from tumours 86 and 106 were
similar instructure to early embryoid bodies from tumour 17, and
their structure has notso far changed with time. However, some
early embryoid bodies of tumour 106appeared to have a more distinct
outer layer of flattened cells (Fig. 1C), resem-bling endoderm.
This outer layer was very distinct in some later embryoid bodiesof
tumour 86, with some of the inner cells organized into stuctures
resemblingearly ectoderm (Fig. 1D). Solid tumours arising after
subcutaneous injection ofboth 86 and 106 embryoid bodies containing
only ECC and nervous tissue.
Embryoid bodies arising from tumour 145 consisted of masses of
ECC, oftensurrounded by a layer of flattened cells (Fig. 1E).
Subcutaneous injection ofthese embryoid bodies gave tumours
consisting mainly of ECC, together withneuro-ectodermal tubules and
simple cuboidal epithelium; these tumoursresembled the subline of
tumour 145 in which differentiative capacity wasrestricted.
DISCUSSION
1. Induction of teratomas
C3H embryos form teratomas more frequently as they are
transferred to thetestis at progressively later stages of
development up to the 8th day. The propor-tions forming teratomas
are: no one-cell eggs (lies et al. 1975), one-third of blast-ocysts
and 7th day egg-cylinders, and two-thirds of 8th day egg-cylinders.
Severalfactors 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 develop-ment 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 ecto-placental cone from 8th day embryos, and
these embryos grow more frequently.
It is known that the majority of blastocysts transferred to
extra-uterine sitesstart 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 73few of these
growths develop into teratomas. The culture of blastocystsincreases
cell number and disorganizes tissue relationships as the
trophoblastlayer spreads out on the substrate: it was therefore
hoped that a higher fre-quency of teratoma formation would be
obtained from cultured blastocysts.Such an increase would be of
value in the induction of teratomas from partheno-genetic
blastocysts (lies et al. 1975) which rarely go on to form
egg-cylinders invivo. Culture for 4 days in a medium did not raise
the incidence of teratoma for-mation by C3H blastocysts above the
original one-third, but it increased theincidence with 129/J
blastocysts from 10 % (Stevens, 1970, and this study) to30 %;
enzymic pre-treatment lowered the incidence of tumour formation
fromboth C3H and 129/J blastocysts cultured for 4 days in a medium,
while longerperiods of culture greatly reduced tumour formation in
both strains. This effectof time is not understood, because embryos
of the same total age can formbenign teratomas (Damjanov et al.
1971a). Tumours derived from blastocystscultured for 4 or 5 days do
not contain significant amounts of ECC, but they doshow a wide
range of differentiated tissues.
2. Development of teratocarcinomas and embryoid bodies
In this study, teratocarcinomas (transplantable teratomas) were
obtained fromC3H blastocysts, 7th and 8th day egg-cylinders;
similar tumours have beenobtained from C3H 8th day egg-cylinders by
Damjanov et al. (1971 b) and from129/J blastocysts and 7th day
embryos by Stevens (1970). Both spontaneousand embryo-derived
teratocarcinomas may either retain the ability to differen-tiate
into many tissues (pluripotent) or their differentiation may be
restricted toone or a few tissues (e.g. Stevens, 1958, 1970;
Damjanov et al. 19716). Thehistology of these C3H teratocarcinomas
on successive transplant generationsshows that 17 retains
pluripotency while 86,106 and 145 have become restricted.
The restriction of developmental potential of some tumours may
be due tothe selection of rapidly dividing ECC whose capacity to
differentiate has be-come restricted: such restrictions may be
associated with abnormal karyotypes(see following paper by lies
& Evans, 1977). It is important to discover
whetherteratocarcinomas maintained as embryoid bodies are more or
less restrictedthan those maintained as solid tumours. This paper
is the first published descrip-tion of embryoid bodies derived from
C3H teratocarcinomas; their structure iscomparable with the simpler
of the embryoid bodies in OTT 6050 ascites fluiddescribed by
Stevens (1970).
It has been shown here that the developmental capacity cf a
teratocarcinomais generally similar whether it is maintained as
embryoid bodies or as a solidtumour. Embryoid bodies of 86,106 and
145 all gave subcutaneous tumoursconsisting of embryonal carcinoma
and nervous tissue; these tumours stronglyresembled the final state
of the solid tumour transplants. For 86 and 106, thisrestriction in
developmental capacity could be related to the observation
thatsolid 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 theircapacity to differentiate (see Results); solid
tumours of 145 became restricted (inone subline) one generation
later than the embryoid bodies first appeared.
Tumour 17 continued to differentiate well, and some solid
implants found onthe wall of the peritoneum after injection of
embryoid bodies developed a widerange of differentiated tissues.
However, the embryoid bodies of 17 have recentlybecome 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 carci-nomas when injected subcutaneously. A
similar progression to yolk-sac carci-noma has been reported with
strain 129/J embryoid bodies (Stevens, 1959;Pierce & Dixon,
1959).
The conclusion from these observations is that tumours may
become restrictedin developmental capacity whether they are
maintained as solid tumours or asembryoid bodies. Both growth
conditions seem to select for cells with rapidgrowth which may have
restricted developmental capacities.
I should thank the following people: Dr C. F. Graham, for his
advice and constant en-couragement, Dr D. Solter, for reading the
manuscript, and S. R. Bramwell, for experttechnical assistance.
This work was supported by the Cancer Research Campaign and
partlyby a Mary Goodger Scholarship.
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(Received 22 April 1976, revised 21 October 1976)