/. Embryol. exp. Morph. Vol. 54, pp. 277-295, 1979
Printed in Great Britain © Company of Biologists Limited 1980
277
Growth and differentiation of an embryonal
carcinoma cell line (C145b)
By Y. E. PAPAIOANNOU, 2 E. P. EVANS,2 R. L. GARDNER 2
AND C. F. GRAHAM 1
From the Department of Zoology and
the Sir William Dunn School of Pathology, University of Oxford
SUMMARY
Several cell and tumour lines were isolated from a single-embryo-derived teratocarcinoma
and their karyotypes and differentiation in adult hosts recorded. The majority of cells
contained normal karyotypes by banding. The cells were injected into blastocysts and
although they sometimes colonized the yolk sac, they never colonized the embryo. Thus
the possession of a normal karyotype is not a sufficient condition for embryo colonization.
The loss of growth capacity was investigated by studying differentiation and tumourigenicity
in a variety of circumstances. The change in appearance from an EC cell morphology to a
big flat cell in culture leads to retardation of growth in adult hosts. When EC cells are
injected into a blastocyst, the ability to grow progressively both in culture and in adult hosts
is lost.
INTRODUCTION
Transplantable teratocarcinomas can frequently be obtained by grafting
normal mouse embryos, at particular stages of embryogenesis, into extrauterine sites (e.g. Stevens, 1967, 1970; Solter, Skreb & Damjanov, 1970; lies,
1977). These tumours contain embryonal carcinoma (EC) cells, grow
continuously, and eventually kill their host. Single EC cells form embryoid
bodies, the ascites form of teratocarcinoma, can differentiate into a wide range
of tissue types in the animal, and they can also form transplantable teratocarcinomas. Thus they can be the sole stem cells of teratocarcinomas (Kleinsmith
& Pierce, 1964).
There are several reasons for thinking that the differentiated cells derived from
EC cells cannot form transplantable teratocarcinomas. For example, the
differentiated cells which form on embryoid bodies are unable to form transplantable growths (Pierce, Dixon & Verney, 1960; Pierce, 1974). EC cells also
differentiate into a wide range of tissue types when injected into the blastocyst
and, with the exceptions mentioned below, the chimaeric animals rarely develop
1
Author's address: Department of Zoology, University of Oxford, South Parks Road,
Oxford, 0X1 3PS. U.K.
2
Authors' address: Sir William Dunn School of Pathology, University of Oxford, South
Parks Road, Oxford, 0X1 3RE, U.K.
278
V. E. PAPAIOANNOU AND OTHERS
tumours from the differentiated cells (Brinster, 1974; Mintz & Illmensee, 1975;
Papaioannou, McBurney, Gardner & Evans, 1975; Illmensee & Mintz, 1976;
Dewey, Martin, Martin & Mintz, 1977; Papaioannou et al. 1978).
Despite this apparent antagonism between differentiation and transplantable
growth, a number of cell lines have been isolated from EC cells which express
differentiated functions but still grow in adults; these include fibrosarcomas and
parietal endoderm carcinomas (Evans, 1972; Martin & Evans, 1974; Lehman,
Speers, Swartzendruber & Pierce, 1974). In addition, fibrosarcomas and
rhabdomyosarcomas have been formed from one line of EC cells injected into
blastocysts (Papaioannou et al. 1978). There is therefore a reasonable doubt
that the ability to grow in a host is necessarily lost as EC cells differentiate.
We have investigated this problem by studying the growth and differentiation
of an embryo-derived EC cell line following injection into the blastocyst,
transplantation into adults, and culture in dishes. Chromosomal banding
studies indicate that this line had a normal female mouse karyotype during
most of these procedures and was free of trisomies and the chromosomal
rearrangements present in most other EC cell lines.
MATERIALS AND METHODS
Tumour derivation and cell isolation
Tumour C3H 145 was derived from an egg cylinder on the seventh day of
gestation (first day of gestation was day of copulation plug), during December
1974 (lies, 1977). The tumour produced several rapidly dividing sublines which
were chromosomally abnormal during subcutaneous transplantation (lies &
Evans, 1977; McBurney & Adamson, 1976). The main line of the tumour was
chromosomally normal and EC cell cultures were obtained from the sixth
transplant generation in February 1976. The tumour was washed in phosphatebuffered saline (PBS, solution A of Dulbecco & Vogt, 1954) and the softer parts
were teased into small fragments with fine forceps. Single cells and small lumps
(containing up to 200 cells) were quickly plated on feeder cells in alpha medium
(Stanners, Eliceira & Green, 1971) lacking nucleosides and deoxynucleosides,
and containing 10% (v/v) heat-inactivated foetal calf serum (FCS) and 15%
(v/v) heat-inactivated calf serum (Flow Laboratories, Irvine, Scotland). The
dishes of feeder cells were prepared as follows: 50 mm diameter plastic tissue
culture dishes (Sterilin Ltd., Richmond, Surrey) were covered with water
containing 1 % (w/v) gelatin (swine skin, type 1; Sigma Chemical Company,
U.K.) and left at 5 °C for 1 h. Subsequently the gelatin was decanted, the
dishes air-dried and covered to confluence with STO cells (Ware & Axelard,
1972) which had been previously treated with mitomycin C to prevent growth
(Sigma Chemical Company; techniques described by Martin & Evans, 1975;
McBurney, 1976).
The medium was changed every 2 days and after 10 days colonies of EC cells
Growth of an embryonal carcinoma cell line
279
could be recognized because the individual cells had characteristically small size,
rapid growth, and clear nucleoplasm. The colonies were sucked up in a micropipette and broken into smaller lumps which were distributed on fresh feeder
cells. Ten days later this procedure was repeated and after a further 10 days the
dishes contained many EC cells which could be subcultured with a 2 min
treatment with 0-125% (w/v) trypsin (Difco Limited, U.K.) and 0-2 rriM
EDTA in PBS. They were maintained in alpha medium with 10% FCS by
subsequent subculture every 2-7 days with the replacement of the medium every
2 days. A variety of cell lines were isolated in this way.
In addition, experiments were conducted on an ascites line of the tumour
which grew as aggregated EC cells. These EC cell aggregates (b4, see Results)
were removed directly from the peritoneum, washed in PBS and suspended in
culture medium until use. The thioguanine resistant clone, C145bTGl, was
derived from C145b4, and it was maintained as an EC cell population by
subculture every 4 days with plating at 2 x 105 cells per gelatin coated, feeder
covered, 90 mm diameter dish. To promote differentiation into big flat cells,
the cells were dissociated and plated at 1 x 106 cells per 90 mm diameter gelatin
coated dish in alpha medium with 10% (v/v) FCS.
Injection into adults
The cells were dissociated into single cell suspensions (90 % or more single
cells) with trypsin, versene and chick plasma (Bernstine, Hooper, Grandchamp
& Ephrussi, 1973), rinsed in tissue culture medium, and resuspended in PBS.
The cells were counted with a haemocytometer and diluted so that 0-25 ml
contained the appropriate number of cells. These were injected subcutaneously
in the back about 2 cm posterior to the nape of the neck. Following injection,
this region was palpated twice a week to time the first appearance of tumour
growths.
Injection into blastocysts
EC cells from culture and from the ascites tumour were injected into blastocysts. The host blastocysts were either from random bred stocks (CFLP,
Anglia Laboratories Limited; PO, Pathology, Oxford) or inbred strains
129J/SV and A2G. The blastocysts were either flushed from uteri on the fourth
day of gestation or morulae were recovered on the third day and cultured
overnight to the blastocyst stage in Whitten's medium (1971). Injection procedures were those of Gardner (1978a) and the microsurgery medium was
either PB1 with 10% (v/v) FCS replacing bovine serum albumin (Whittingham
& Wales, 1969) or the previously described alpha medium with 10% FCS.
The EC cells were prepared for microsurgery in a variety of ways. EC cell
colonies and ascites aggregates were picked with a micropipette, dissociated in
standard EDTA/trypsin or in 0-5 mM EGTA in PBS, and replaced in culture
medium. Between two and ten of these cells were injected. In one series of
280
V. E. P A P A I O A N N O U AND OTHERS
Fig. 1. Four blastocysts l h after injection of C145 b4 ascites aggregates. The
aggregate is obvious as a loose cell clump in three and as a compact body in the
fourth. JCM, inner cell mass; ag, ascites cell aggregate. Arrows indicate the healed
injection sites.
experiments EC cells were aggregated with immunosurgically isolated inner cell
masses (ICMs; Solter & Knowles, 1975) and cultured for 3 h before injection
into blastocysts. Intact ascites aggregates were also injected. These were
selected on the basis of size. The number of cells in similarly sized aggregates
were counted by the following procedure. They were rinsed with PBS and
placed on a clean slide in a small drop. A coverslip was dropped over the
aggregate and the cells fixed by running a 3:1 (v/v) ethanol:acetic acid solution
under the coverslip. The nuclei were then stained by running a solution of
1 % (w/v) orcein in lactic acid under the coverslip. During these procedures, the
cells were observed under the microscope to establish that none were lost. The
average cell number found in a sample of 14 aggregates was 52 cells with a
range of 33-82 cells. All the injected blastocysts were cultured for at least 1 h
until they had healed and reexpanded. The injected cells could often be seen
attached to the ICM and aggregates were prominent in the blastocoel (Fig. 1).
The injected blastocysts were surgically transferred to the uteri of CFLP
foster mothers on the afternoon of the third day following mating to a
vasectomized male. Some of the operated embryos were analysed at mid-
Growth of an embryonal carcinoma cell line
281
gestation and others were left to term. At midgestation the embryos were
dissected from the uterus on the 12th—15th day, the visceral yolk sac was
separated from the foetus and both were analysed for glucose phosphate
isomerase (GPI) by starch gel electrophoresis. About 2 % of the minor component can be detected by this assay. C145 cells are homozygous for the
Gpi-lb allele at the Gpi-1 locus while all the host strains are homozygous for the
Gpi-la allele. A second marker, pigment, was also used for all the embryos that
were allowed to proceed to term. In this case the host strains are all albino
whereas C145 cells are genetically pigmented. All animals born were thus
scored for coat and eye pigmentation at birth or shortly thereafter. Some were
test bred to detect a possible contribution of the C145 cells to the germ cells;
many were also bled and their internal organs were sampled for GPI analysis
when they were sacrificed as adults.
Transplantation to the kidney
Single ascites aggregates and aggregates contained in blastocysts were transferred beneath the left kidney capsule using a mouth controlled flame polished
micropipette. The recipients were male C3H mice which are therefore syngeneic
with the C145b cells.
Preparation of karyotypes
The culture medium was replaced with Eagle's minimal essential medium
(MEM, Flow Laboratories) without serum and the cells both removed off the
bottom of the dish and partially disaggregated by pipetting; ascites aggregates
were washed three times in MEM and then also disaggregated by pipetting.
Standard air-dried preparations were made from these cell suspensions by the
method of Ford (1966), modified by using aqueous 0-56% (w/v) KC1 as the
hypotonic solution. Additional preparations were made from whole aggregates
by'Ltreating them with hypotonic solution, fixing, and finally disaggregating them
on'slides with aqueous 60 % (v/v) acetic acid, as described for foetal embryonic
membranes (Evans, Burtenshaw & Ford, 1972).
To score chromosome numbers, the slides were immediately stained with
toluidine blue (Breckon & Evans, 1969). To band chromosomes, the metaphase
spreads on the slides were 'aged' at room temperature for at least 1 week.
The karyotypes were G-banded by a modification of the method described by
Gallimore & Richardson (1973). The banded metaphases were first analysed
by eye and then selected metaphases were photographed and the chromosomes
arranged according to the standard nomenclature proposed by Nesbitt &
Francke (1973).
282
V. E. PAPAIOANNOU AND OTHERS
Histology
Growths were fixed, sectioned and stained as previously described (lies et al.
1975). In the case of transplants beneath the kidney capsule the whole growth
was sectioned and every section studied. In the case of large subcutaneous
tumours a sampling procedure was used: between one-tenth and one quarter of
the growth was sectioned and every tenth section studied. All kidneys were
weighed within 5 min of dissection.
RESULTS
We investigated the growth and differentiation of the derivatives of C145b in
several situations. First, several types of cell and tumour lines were isolated and
their karyotypes and differentiation in adult hosts were recorded. These cell
lines were then injected into blastocysts and although they sometimes colonized
the yolk sac, they never colonized the embryo. This loss of growth capacity by
the cells was investigated further by studying tumourigenicity in extra-uterine
sites and by taking an ascites-derived cell clone through differentiation in cell
culture.
Isolation and karyotypes of EC cell lines and ascites cell aggregates
We studied the relationship between the karyotype and the ability of cells to
grow in culture and to form ascites tumours in the animal. Previous studies had
shown that at the fourth transplant generation a subline of the tumour developed multiple chromosomal rearrangements, formed an ascites tumour (lies &
Evans, 1977), and grew well in culture (called C145a; McBurney & Adamson,
1976). The chromosomally normal main line of the tumour did not grow well in
culture until the sixth transplant generation (called C145b).
bl cell line. A cell line of EC morphology was isolated 2 months after placing
a sixth generation tumour in culture on feeder cells. The tumour from which it
was isolated contained glandular, ciliated, and keratinized epithelia, nerve, and
EC cells. The tumour had a normal female karyotype which was also retained
in cell culture (Fig. 2a; Table 1). These cells were used for blastocyst injection
within 15 days of thawing, and therefore the majority probably had a normal
karyotype.
b2 cell line. After 3 months in culture, bl was injected under the skin and into
the peritoneum. One month later a subcutaneous tumour developed and it was
cultured without feeder cells. This line had a normal female karyotype (Table 1)
and formed a subcutaneous tumour on reinjection to an adult host; this tumour
contained ciliated, glandular, and keratinized epithelia, loose connective tissue,
hyaline cartilage, smooth and striated muscle, nerve and EC cells. Cells from
this line were injected into blastocysts within 1 month of the karyotype analysis.
b3 cell line. The tumour from b2 was retransplanted under the skin and into
C145b TGI
b2
b3
b4
bl
Cell line
In vivo
Tumour of origin
One month culture
Frozen, thawed, 2 months culture
One month culture
One month culture
Two weeks culture
Three weeks culture after cloning
+
In vivo
+
—
—
+
without ( - )
feeders
Time of karyotype
preparation
or
Grown
with(+)
i
1
2
—
—
—
1
1
38
40
38
25
25
22
21
44
43
39
—
2
—
2
1
3
5
A.
1
1
—
—
2
2
1
41
42
—
—
—
1
—
—
—
Number of metaphases with
particular chromosome counts
Table 1. Karyotypes of cell lines and ascites cell aggregates
5
4
3
7
5
5
8
4's
4's
4's
None
None
None
None
No. of
karyo- Abnormal
types chromobanded somes
oo
1
|
I
TO
I
284
II
V. E. PAPAIOANNOU AND OTHERS
II it II
II II II M
It
H >•
(a)
Fig. 2. Karyotypes of C145. (a) A normal female karyotype obtained from the
C145 bl cell line, (b) An abnormal female karyotype obtained from the C145 b3
cell line. This shows the rearrangement in both chromosomes 4. In the chromosome 4
on the right, two additional major positive bands have been inserted into the C6
band region. In the chromosome 4 on the left, the segment between bands C6 and D3
appeared to be inverted. These rearrangements were later found in both C145 b4
and C145b TGI.
the peritoneum and the subcutaneous tumour which developed was grown in
culture without feeders. The chromosome counts were normal but banding
studies on five karyotypes showed that both chromosomes 4 were rearranged
while the rest of the karyotype was that of a normal female (Fig. 2b). In one
chromosome 4, two additional major positive bands were inserted into the C6
(Nesbitt & Francke, 1973) band region giving an approximately 10% increase
in total chromosome length over normal. The length of the other chromosome 4
was normal but the segment between bands C6 and D3 appeared to be inverted.
Cells from this line were injected into blastocysts within 1 month of the karyotype
analysis.
Growth of an embryonal carcinoma cell line
285
ft
M it II
M II II
li
II
(b)
Fig. 2{b). For legend see opposite.
b4 ascites tumour. After three transplant generations, at each of which the
cells were injected into the peritoneum and under the skin, the tumour from b2
formed an ascites tumour with aggregates which lacked an obvious rind of
endoderm. These aggregates had normal chromosome numbers (Table 1)
but analysis of five banded karyotypes showed that both chromosomes 4 were
rearranged in the same way as those of b3. During passage as an ascites tumour,
solid tumours also formed on organs in the peritoneum. Two tumours were
examined and these were poorly differentiated and contained EC cells with a
little loose connective tissue. Cells from b4 aggregates were injected into
blastocysts within 5 months of the karyotype analysis.
C145b TGI. After five transplant generations over 5 months, b4 aggregates
were placed on feeder cells and grown in the presence of 5 /tg/ml thioguanine.
A colony was picked which grew well on feeder cells in the presence of 15 /tg/ml
thioguanine. This colony was assumed to be derived from a single cell (a clone).
19-2
286
V. E. PAPAIOANNOU AND OTHERS
When injected into adult mice it formed a tumour with EC cells and some loose
connective tissue. This clone had normal chromosome numbers but both
chromosomes 4 were rearranged as previously described.
These results show that, as C145b cells adapted to or were selected for rapid
growth, there were minor rearrangements in the karyotype. Up to the time of
isolation of bl, the tumour was slowly dividing and was on average passaged
every 2\ months; after the appearance of minor chromosomal rearrangements
(b3) and the conversion of the tumour to ascites form (b4) it had to be passaged
every month on average. The range of differentiated cell types found in samples
of the tumour decreased from a wide range (bl and b2) to a restricted range
(b4 and C145b TGI) after the appearance of minor chromosomal rearrangements.
Transplantation to the blastocyst
We transplanted cells from C145b to the blastocyst at different times during
its progression from a feeder dependent cell line (bl), to a feeder independent
cell line (b2, b3), to an ascites line without a morphologically distinct endoderm
layer (b4). The cells were pretreated in several ways before injection (see
Materials and Methods, Table 2). The intention was to discover a relationship
between the characters of the lines and colonization of the embryo.
The results of all the injection experiments are in Table 2. In total 605 animals
were analysed for a contribution of the injected C145b cells and in 397 of these
the visceral yolk sac was also analysed for the contribution of C145b. A contribution of the injected cells was only found in five animals and in each the
C145b cells had only contributed to the visceral yolk sac. All but one of the
chimaeras was obtained from a cell line which was chromosomally aberrant:
one was from b2, one from b3, and three were from line b4. In spite of breeding
to study the possible contribution of C145b to the germ line (68 animals bred
and over 4000 offspring observed for coat colour), and a survey of adult tissues
by GPI analysis (91 animals), no chimaeras were detected among the animals
which developed into adults.
Growth of ascites aggregates alone and inside blastocysts
To ascertain whether the capacity for growth was lost soon after the transfer
of ascites aggregates into the blastocyst, we compared the growth of aggregates,
blastocysts, and blastocysts injected with aggregates.
Culture. Twenty-two single aggregates were placed in separate Petri dishes.
The majority formed big flat cells but eight grew into confluent EC cell cultures
during the next three weeks. Each of these were injected subcutaneously and
each formed a tumour which contained EC cells with a little connective tissue.
Thirty-four aggregates were injected into CFLP blastocysts and cultured under
identical conditions. Thirty of these formed only giant cells and ICM-like cells
during the next fortnight and their behaviour was indistinguishable from that of
CFLP
CFLP
129
CFLP
A2G
129
CFLP
PO
A2G
129
28
4
21
2
10
1
4
18
1
10
26
125
CFLP
59/62
126/185
657/862
3/7
24/32
95/125
8/8
150/196
15/30
125/155
10/11
42/51
7
23
90$
0
45§
85
404
42J
20
15
85
7
36
201
129
15
21
26
21
60
10
0
GPI
GPI
93
12
11
Internal
organs
Blood
Term
10
51
7
0
Test
breeding
> 200
518
0
3410
Total no.
offspring
from test
breeders
N
ag, ascites cell aggregates.
* Refers to the number of animals born or to the number of implantation sites if foster mothers were sacrificed at midgestation. Numbers
exclude foster mothers that did not become pregnant,
t In seven cases the yolk sac was not GPI typed.
t Including one yolk-sac chimaera.
§ Including three yolk-sac chimaeras.
bl clumps or cells
clumps
cells
cells
b2 cells+ ICM
cells
cells
b3 cells
cells
b4 whole ag
dissociated ag
Totals
C145 subline
No. of
foster
mothers
Host
blastocyst
strain
No. animals
recovered* Foetus and Coat and
eye
No. embryos
yolk sac
transferred
pigment
GPIt
Midgestation
Numbers analysed
Table 2. Summary of the results of injection of C145 into blastocysts showing the numbers of animals analysed
by GPI at midgestation and by GPI, pigment, and test breeding at term or as adults
to
oo
*^'
*^
O
""*
Ci
a
§
f1
288
V. E. PAPAIOANNOU AND OTHERS
I
20
I
I
I
•
« I t t t *
(b)
" • "
300
,;=18
o
8
Kmbryoid bodies alone
1
.
m
Embryoid bodies
in blastocysts
^L
(c)
/i = 10
Blastocysts alone
(d)
H=10
Controls
CD
003
001
+
+
+
+
+
+
+
001 0-03 005 007
0-15 0-25 0-35
Wt. left kidney minus wt. right kidney (g)
+
0-45 0-55
Fig. 3. Weight changes following injection of cells under the kidney capsule. In all
experiments cells were injected under the left kidney capsule of C3H mice. After a
month, the host was killed and both kidneys in each animal were weighed. The
weight of the control right kidney was then subtracted from that of the injected left
kidney. In unoperated controls (line d), it was found that the left kidney always
weighed less than the right kidney (average difference equals —0-016 g). Similarly,
nine out of the ten left kidneys which had been injected with CFLP blastocysts alone
(line c) weighed less than the right kidneys (average difference equals —0-014 g).
14 out of 18 left kidneys which had been injected with CFLP blastocysts containing
b4 aggregates (line b) weighed less than the right kidneys (average difference
equals +0023 g). In three cases (closed circles) EC cells were found in the left
kidney. EC cells were found in all the kidneys injected with aggregates alone
(line a); 16 injected left kidneys weighed more than the right kidneys (average
difference equals +0129 g). For clarity the weight differences are grouped in this
figure: the weights on the left scale fall within 001 g of the indicated weight and
those on the right scale fall within 005 g of the indicated weight. The weight increase
in left kidneys containing tumours may be due both to the weight of the tumour and
to the presence of haemorrhage.
six control uninjected blastocysts. Four gave EC cell outgrowths whose tumourigenicity was not tested. The proportion of aggregates forming EC cell cultures
was significantly greater when they were not contained in blastocysts (P < 0-05,
X on a 2 x 2 contingency table with Yates correction for small samples.
Bailey, 1959). We believe that this difference in growth behaviour is a real effect
of the blastocyst, because in two of the cases where EC cells grew out from
injected blastocysts it was noticed that the aggregate was protruding from the
injection site at the time of culture and was therefore not restricted by the
blastocyst.
Transplantation beneath the kidney capsule. Twenty aggregates were trans-
Growth of an embryonal carcinoma cell line
Embryonal carcinoma cells
289
Big flat cells
3
[
3-5 X10 . n = 6
501 XIOs,#i = 10
I
7-0 X10 3 ,/; = 8
1-03 X10 4 ,/; =
0
« 8
5-0X10 4 ,/; = 10
4 M
2
o
0
8
3
4
-
-
5 0 X 1 0 4 , / ; = 10
10 X 10s, //= 10
1 0 X 10s,« = 9
|J
0
8
6
4
5-0X10S,H =
i
20
•
i
•
i
•
i
•
i
40 60
80 100
Days after injection
>
5-0X10 5 ,/; = 10
10
i
120
0
20
40
60 80 100. 120
Days after injection
Fig. 4. Appearance of palpable growths after cell injection. Various numbers of
cells were injected subcutaneously and the mice were felt twice a week to score the
time of first appearance of palpable growths. The EC cells (left side) formed palpable
growths faster than the big flat cells (right side).
planted singly beneath the left kidney capsule of recipient mice and left for
1 month. These mice each developed a tumour which contained EC cells with a
little loose connective tissue (16 tumours sectioned and studied). Eighteen
aggregates were injected into CFLP blastocysts and these were then treated as
above. A tumour developed in three cases and contained EC cells (all kidneys
sectioned and studied). In the other cases a small haemorrhage spot was found.
In all these spots, as in the haemorrhages from control blastocyst transplants
(eight sectioned and studied), histological examination revealed cell debris
consisting of degenerate giant cells and in about half the cases small cells were
found in a dense matrix. The proportion of aggregates alone which form EC cell
growth (20/20) is significantly greater than the proportion of aggregates
contained in blastocysts which do so (3/18, P < 0-001, same test). The effect of
the blastocysts on the aggregates growth is apparent when the weight of the
control right kidney is subtracted from the weight of the left injected kidney
(Fig. 3). On average there was an increase of 0-1 g weight in kidneys injected
with aggregates alone when compared with kidneys injected with similar size
aggregates contained in CFLP blastocysts. The failure of the CFLP blastocysts
to form teratocarcinomas or teratomas when transplanted by themselves is
expected; embryos do not develop in this way when transplanted to histoincompatible hosts.
These results show that CFLP blastocysts restrict the growth of EC cells
in culture and in extra-uterine sites. In cell culture, the aggregates which did not
290
V. E. PAPAIOANNOU AND OTHERS
form EC cell growths differentiated into big flat cells and it was possible that the
blastocysts promoted this transition both in culture and in extra-uterine sites.
Differentiation in cell culture and retarded growth in the adult
We investigated the effect of differentiation in cell culture on the ability of
cells to grow in adult hosts. The EC cells from C145b TGI rapidly formed
tumours when 5 x 104 or more cells were in each inoculum (Fig. 4). By 3 days
after plating at low density, these EC cells had formed big flat cells resembling
those produced by the ascites aggregates in cell culture (previous section).
These big flat cells formed detectable growths slower than the EC cells, and at
any particular time after the inoculation of a particular number of cells, a
smaller proportion had formed growths when compared with the EC cells
(Fig. 4). We concluded that the change to a big flat cell phenotype had retarded
the ability of these cells to grow in adults.
Four of the growths obtained from the inoculation of 5 x 105 big flat cells
were fixed and sectioned after 2 months. These growths principally contained
EC cells as did the last tumour which developed at 120 days after the injection
of 5 x 105 big flat cells. We concluded that only C145b TGI cells with an EC cell
morphology could grow rapidly in the adult.
DISCUSSION
Progression of the tumour and cell lines
The progression of this tumour was similar to that previously described
(Stevens, 1970; lies, 1977) and shows a common progression to chromosome
abnormality, rapid growth, ease of cell culture, and a reduction of cell types
formed in solid tumours. This relationship is not absolute, however, since it has
been possible to isolate a pluripotential cell line from b4 which retains its
chromosome abnormality (M. W. McBurney, personal communication).
Our studies support the observation of lies & Evans (1977) that there is no
one chromosome abnormality common to EC cells. C145 formed an ascitic and
a solid tumour with trisomy for chromosome 11, an addition to chromosome 1
and a deletion from chromosome 14 (lies & Evans, 1977), abnormalities that
were well preserved in cell culture (C145a, McBurney & Adamson, 1976). By
contrast C145bl and b2 had normal karyotypes and the only abnormalities in
C145b ascites and solid tumours were rearrangements in both chromosomes 4.
Since rearrangements were only found in the chromosomes 4 at the C6 band,
it is tempting to speculate that they arose as a result of an interchromosomal
rearrangement. If this were so, then the rearrangement must have been complex
because of the banding pattern and the increase in length of one chromosome.
The two extra bands in the longer chromosomes could be reconciled with an
intrachromosomal duplication and inversion of bands C3 and C5 but it is
equally possible that additional material had been inserted from another source.
Growth of an embryonal carcinoma cell line
291
It was therefore not known how chromosomally unbalanced the cell line might
be.
These results demonstrate that sublines can be derived from a single embryoderived teratocarcinoma with no common chromosomal abnormality, and
indeed that a normally banded karyotype is consistent with transplantable
teratocarcinoma growth (these studies and lies & Evans, 1977) and growth in
cell culture.
Colonization of embryo and growth in the kidney
Previous studies have shown that EC cells with both normal and abnormal
karyotypes can colonize the blastocyst. However, it is only those EC cells with
an apparently normal karyotype which have colonized the gonads and also
formed viable gametes (Cronmiller & Mintz, 1978). We hoped that colonization
of the embryo and successful progression through meiosis would occur whenever an EC cell with a normal karyotype was injected into the blastocyst.
In fact the C145b cells colonized in only five instances and in each case they
contributed exclusively to the visceral yolk sac. In four of the five cases, the
C145b cells which did colonize the yolk sac (b3 and b4) had rearranged chromosomes 4 and these observations suggest that the possession of a normal karyotype does not increase the probability that EC cells will colonize the developing
foetus.
The failure of these cells to colonize the embryo may be explained in several
ways. It was possible that only a small subpopulation of the injected cells had
the capacity to grow despite the EC cell appearance of the majority of the cells.
This explanation is unlikely since so many bl, b2 and b3 cells were injected.
In the case of b4 it is excluded since the ascites aggregates failed to colonize the
embryo while identical aggregates were able to grow both in culture and when
injected beneath the kidney capsule (Fig. 3).
Another possibility was that some EC cells did grow in recipient blastocysts,
eventually killing the host blastocyst. This is an unlikely explanation of our
failure to observe colonization because the proportion of recipient blastocysts
which developed normally amongst those transferred to foster mothers which
became pregnant (605/862 or 70%; Table 2) compares favourably with the
proportion of normally developing embryos in transfer studies involving the
injection of normal embryonic cells (Gardner, 19786).
Ascites aggregates lose the capacity to form EC cell growths both in the
kidney (Fig. 3) and in culture after being injected into the blastocyst and so we
favour a third explanation. It is possible that once in the blastocyst they differentiated into slow growing big flat cells, a transition frequently observed in cell
culture. Big flat cells show certain properties in common with the visceral
endoderm cells of the normal embryo (Adamson & Graham, 1979, see below)
and these similarities may extend to their transplantation behaviour. It is
known that primitive endoderm cells only colonize the parietal and visceral
292
V. E. PAPAIOANNOU AND OTHERS
endoderm layers of the yolk sacs when transplanted to a host blastocyst (Gardner
& Papaioannou, 1975; Rossant, Gardner & Alexandre, 1979; Gardner &
Rossant, 1979; R. L. Gardner, personal observations) and any injected cell
which mimicked the phenotype of endoderm cells would thus be excluded from
the foetus. The observation that C145b colonization was only observed in the
visceral yolk sac (we did not test for colonization of the parietal yolk sac)
supports this interpretation. A similar loss of EC cell growth capacity after
being injected into blastocysts is observed with several other EC cell lines which
also fail to colonize the embryo. By contrast, a single cell line which does
colonize the embryo at a high frequency continues to grow in culture after
injection into blastocysts (Papaioannou, 1979).
We are unable to exclude a fourth possibility. It may be that EC cells from
this tumour are adversely affected by some features inside the blastocyst and
that they cease division and die.
Differentiation in cell culture and retarded growth in the adult
It is difficult to observe closely the behaviour of the EC cells inside the blastocyst, but in cell culture it was seen that C145b TGI EC cells readily changed,
into big flat cells as soon as they were plated without feeder cells. A similar
change in appearance occurred at the circumference of C145 b4 aggregates
which attached to tissue culture dishes and also when b2 or b3 were grown at
low density. Similar changes in phenotype have been observed at lower frequency with other EC cell lines (e.g. Martin & Evans, 1974, 1975; Nicolas et al.
1975; Burke, Graham & Lehman, 1978).
The transition to big flat cells has been well studied with other EC cell lines
and clones. The transition is promoted by low density plating and retinoic acid
(Burke et al. 1978; Strickland & Mahdavi, 1978), and it involves the loss of EC
cell characters and the gain of features which are similar to those of visceral
endoderm cells (Adamson & Graham, 1979; Graham, 1979). For two of these
lines, it has also been shown that the slow growth of the big flat cells can be
attributed to a requirement for epidermal growth factor; the addition of this
polypeptide promotes cell division and this observation shows that the big flat
cells are not irreversibly senescent (Rees, Adamson & Graham, 1979). In the
case of C145b TGI, it has been shown that the big flat cells, like all other EC
cell derived big flat cells, synthesize principally type I-like collagen (Adamson,
Gaunt & Graham, 1979) and this observation and the morphology of these cells
makes it reasonable to assume that the properties of these cells are similar to
those of big flat cells produced from other EC cells.
We have shown that the transition to big flat cells in culture retards the
subsequent growth of the cells in an adult host. The retardation of growth is
particularly obvious at 25 days after inoculation, when no big flat cells have
formed palpable growths while many EC cells have done so (Fig. 4). This
Growth of an embryonal carcinoma cell line
293
growth retardation might by itself block the ability of injected cells to colonize
the blastocyst.
We do not know if the change from an EC cell to a big flat cell morphology is
stable. The observation that EC cell growths eventually develop from inocula of
big flat cells does not show that the big flat cell phenotype is unstable because a
few contaminating EC cells may also have been transplanted.
CONCLUSIONS
1. When the EC cell line C145b is injected into blastocysts which are returned
to the uterus, it only colonizes the yolk sac at low frequency.
2. The possession of a karyotype which appears normal by banding studies
is not a sufficient condition for subsequent colonization of the embryo by an
EC cell.
3. The change in appearance from an EC cell morphology to a big flat cell
in culture leads to retardation of growth in adult hosts.
4. When this EC cell line is injected into a blastocyst, its ability to grow
progressively both in culture and in adult hosts is lost.
We would like to thank Helen Mardon and Zillah Deussen for their expert and patient
technical assistance and Joan Brown for preparing the manuscript. The MRC provided
financial support for this work. We also thank Stephen Gaunt for the gift of cell line
C145bTGl.
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{Received 12 March 1979, revised 30 July 1979)