/. Embryol. exp. Morpli. Vol. 44, pp. 93-104, 1978
Printed in Great Britain © Company of Biologists Limited 1978
93
Participation of cultured teratocarcinoma
cells in mouse embryogenesis
By V. E. PAPAIOANNOU, 1 R. L. GARDNER, 1
M. W. McBURNEY, 2 C. BABINET 3 AND M. J. EVANS4
From the Department of Zoology, Oxford
SUMMARY
Mouse blastocysts were microsurgically injected with embryonal carcinoma cells from
in vitro teratocarcinoma cell lines CM, C86, SIKR-OSB, and PCC3/A/1. The embryos were
allowed to develop to term and the resulting offspring were analysed for chimaerism using
coat colour markers and isozyme differences of the enzyme glucose phosphate isomerase.
When injected into blastocysts, cell line C86 produced tumours in six of 74 animals born.
The tumours were detected at birth and were poorly differentiated neuroectodermal teratocarcinomas. Cell line C17 gave 13 chimaeras in 77 mice born, five of which showed chimaerism
only in normal tissues, mainly melanocytes of the coat and eye. The other eight chimaeras
developed tumours. Seven of these developed in adult animals and were mainly fibrosarcomas. Cell line SIKR-OSB resulted in one normal chimaera in 44 mice born. Of 86
animals born following injection of cell line PCC3/A/1, there was one chimaera with a small
tumour and three normal chimaeras. The levels of chimaerism were generally very low. The
mice were test bred but with no evidence of germ line chimaerism. The karyotypes of all the
cell lines were abnormal. How this and other factors such as cell cycle times might affect
the incorporation of embryonal cells into the developing embryo is discussed.
INTRODUCTION
Mouse embryos of various developmental stages can form teratocarcinomas
when grafted to ectopic sites in adult hosts (Stevens, 1968, 1970; Solter, Skreb
& Damjanov, 1970). Stem cells of these tumours, known as embryonal carcinoma cells (EC cells), can be grown in culture and can produce a variety of
differentiated cell types when injected into adult hosts (Kleinsmith & Pierce,
1964). To discover whether similar factors control both the differentiation of
EC cells and those of the normal embryo, these cultured cells were restored to
the environment of the early embryo by microsurgery (Papaioannou, McBurney,
Gardner & Evans, 1975). Participation of EC cells in normal morphogenesis
was observed. We now present the completed analysis of the animals from
Authors' addresses:
1
Department of Zoology, South Parks Road, Oxford, OX1 3PS, UK.
2
Department of Biology, University of Ottawa, Ottawa, K1N6N5, Canada.
3
Institute Pasteur, 25-28 Rue du Docteur-Roux, 75724 Paris-Cedex 15, France.
4
Department of Anatomy and Embryology, University College London, Gower Street,
London, WC2, UK.
7
KMB
44
94
V. E. PAPAIOANNOU AND OTHERS
experiments with three cell lines and, in addition, the results of injection of a
fourth in vitro cell line. Several more chimaeric animals have been detected,
notably through the growth of tumours in adulthood. Cultured cell lines have
been used with the intent of eventually isolating mutant EC cells and introducing these mutants into mouse stocks via the germ line.
MATERIALS AND METHODS
Cell lines
The embryonal carcinoma cell lines C86 and C17 derived from teratocarcinomas of mouse strain C3H and the line SIKR-OSB derived from strain
129 have been described (Evans, 1972; McBurney, 1976; Papaioannou et al.
1975; lies & Evans, 1977). In addition, a fourth EC cell line PCC3/A/1 (Jakob
et al. 1973; Guenet, Jakob, Nicolas & Jacob, 1974) derived from the tumour
OTT6050 from a 6-day 129-strain embryo (Stevens, 1970) was injected into
blastocysts of the strain AG/Cam. PCC3/A/1 has 40 chromosomes and is XO
(Nicolas et al. 1976). It has been in culture for over 300 generations and is
genetically non-albino, white-bellied agouti, and homozygous for the a allele
of the glucose phosphate isomerase locus (Gpi-1).
Microsurgery
Injection of mechanically isolated clumps of PCC3/A/1 embryonal carcinoma
into blastocysts was carried out as described for the other cell lines (Papaioannou et al. 1975). Additional experiments were also carried out in which 1-5
isolated PCC3/A/1 cells or C17 cells were injected directly into the inner cell
masses of a number of blastocysts of the appropriate genotype. The PCC3/A/1
cells were mechanically dissociated; C17 cells were disaggregated in 0-5%
trypsin. Injected embryos were transferred either to the oviduct or to the
uterus of random bred CFLP females (Anglia Laboratory Animals Ltd) in the
first or third day of pseudopregnancy respectively and allowed to develop to
term.
Assessment of chimaerism
The offspring were inspected frequently for ocular and coat colour chimaerism evidenced by black pigment in the albino CFLP hosts or agouti hairs
in the extreme non-agouti AG/Cam hosts. Blood samples were taken at weaning
for glucose phosphate isomerase (GPI) horizontal starch gel electrophoresis.
All mice that reached breeding age were bred with mice from the same experiment or with mice from the strain of the host blastocyst to test for functional
gametes of donor origin. This should yield pigmented offspring in experiments
using C3H EC cells and white-bellied agouti offspring in experiments with 129
EC cells. However, since none of the chimaeras from cell line C86 were pigmented, it was thought possible that this cell line had lost the capacity to form
pigment. Hence, test offspring from all animals injected with C86 were also
Cultured teratocarcinoma
cells in mouse embryogenesis
95
Table 1. Rate of development and chimaerism of blastocysts injected with
embryonal carcinoma cells or cell clumps
EC
cells
injected
C86 clumps
C17 clumps
C17 cells
SIKR-OSB clumps
PCC3/A/1 clumps
PPC3/A/1 cells
Host
No.
Mice born/
blastocyst pregnant embryos Development
No.
Chimaeric
strain
recipients transferred
(%)*
chimaeras
CFLP
CFLP
CFLP
AG/Cam
AG/Cam
AG/Cam
15
7
8
9
19
4
74/105
43/71
34/46
44/65
68/127
18/28
70
60
74
68
54
64
6
5
8
1
4
0
8
12
24
—
6
Excluding recipients that failed to become pregnant.
typed for GPI. Finally, at autopsy of the animals derived from blastocysts
injected with EC cells, samples of tissues, internal organs, and any tumours
detected were taken for GPI analysis and histology. Tumours were sectioned at
8-12/tm and alternate slides were stained with haematoxylin and eosin and
Masson's trichrome. A small proportion of the animals were lost, decomposed
or were otherwise unavailable for this analysis.
RESULTS
The experiments are summarized in Table 1. While the rate of development
of injected blastocysts is similar for all cell lines, C17 produced the highest rate
of chimaerism. The results of injection of each cell line can be individually
summarized as follows:
C86
The six chimaeras resulting from injection of clumps of C86 stem cells have
been described (Papaioannou et al. 1975). These six mice had no pigment in
skin or eyes but they all developed poorly differentiated tumours (Fig. 1) at or
shortly after birth and were also chimaeric in at least one normal tissue as
judged by GPI. None reached breeding age. Of the other 68 offspring there is
no information on three animals. The rest were scored negative for pigment
cell and GPI chimaerism. Of the 57 mice that reached breeding age, 49 were
fertile and were test bred. A total of 3376 progeny were scored, an average of
69 offspring per mouse (range 4-179), with no evidence of germ line chimaerism.
The majority of experimental animals were killed at about one year of age; no
further tumours or other abnormalities were detected.
C17
Thirteen chimaeric mice resulted from injection of C17 cells or cell clumps
into blastocysts (Table 1). The 64 non-chimaeric mice were all scored negative
7-2
96
V. E. PAPAIOANNOU AND OTHERS
100//m
Fig. 1. Poorly differentiated tumour from mouse T33.
for pigment and of the 57 that reached breeding age, 47 as well as 5 of the
chimaeras, were fertile when test bred. In all 2889 offspring were scored (average
56/mouse, range 8-176) with no evidence of germ line chimaerism. Most of
the non-chimaeric animals were killed at about one year of age and their
internal organs were analysed for GPI. The number of progeny as well as the
extent of chimaerism is detailed for the 13 chimaeras in Table 2. Chimaeras T6,
T84, T85 and T89 were reported in the preliminary paper (Papaioannou et al.
1975) but the last three were still living at the time of publication. Of the 13
C17 chimaeras, five were normal tumour-free mice but with low levels of eye
and/or coat pigmentation. Pigmented melanocytes were seen in both the
retinal pigmented epithelium and in the choroid layer of the eye as illustrated
in Fig. 2. T84 was the only one of these normal chimaeras to show internal
chimaerism by GPI analysis. The other eight chimaeras all developed tumours.
Mouse T6 was the only one to develop tumours shortly after birth; the others
developed rapidly growing tumours when they were over 3 months of age and
were killed within several weeks of the initial detection of the tumours. GPI
analysis indicated that these tumours were largely composed of progeny of the
injected embryonal carcinoma cells. Most of these mice showed little or no
evidence of embryonal carcinoma-derived cells in normal tissues. T89 and T96
were negative for blood chimaerism at weaning but at death had trace amounts
of EC type GPI in serum but not in packed red cells. This is perhaps due to the
Cultured teratocarcinoma
cells in mouse embryogenesis
97
Table 2. Summary of chimaeric animals following injection of C17 cells
or cell clumps. Pie diagrams indicate approximate GPI contribution of
C17 derived cells {black)
Chimaerism
bO
o. of offs;pri
oat
0
K
16
0
K
25
74
OOOO OOOOOO
OOOO OOOOOO
OOOO OOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOO ©OO3OO*'
T84
°
Clump
K
T85
?
?
Clump
Cells
T127
T6
?
2
Cells
K
3
0
Cells
K
46
74
Clump
fd
1
0
umour n
umour
pleen
luscle
ung
iver
idney
onads
eart
rain
lood
d
yes
ge (wks)
9
ate
ijection
himaera no.
U W O Q o a U I ^ - J - l ^ o n H I -
on
T129
GPI
Z
<
<J
T122
Pigment
c
* 2
TI31
Of
T89
Clump
K
14
0
d
d
d
d
2
d
Cells
K
26
54
Cells
K
19
0
Cells
fd
24
74
Clump
K
20
0
Cells
K
22
17
Cells
K
19
OOOO
OOOOOO*'
33
TI36
TI34
T96
T128
T135
OOOO OOOOOO*
000000000003
OOO
3
000000000003
000000000003
000000000003
K = killed; fd = found dead; open circles = non-chimaeric.
presence of the waste products from the large tumours. T134 also had EC type
GPI in blood at death but not at weaning, in this case possibly attributable to
post-mortem changes since the animal was found partially decomposed.
SIKR-OSB
The single chimaera resulting from injection of clumps of this cell has been
described (Papaioannou et ah 1975). Of the other 43 mice born seven were
cannibalized at birth, the remaining 36 were negative for internal GPI chimaerism. A total of 406 offspring were scored from 11 mice test bred (average
37/mouse, range 5-66). There were no germ line chimaeras.
98
Y. E. PAPAIOANNOU AND OTHERS
50 urn
Fig. 2. Pigment in the eyes of mouse T89. (A) Flat mount of a choroid with areas
of pigmentation evident. (B) Detail of choroid and retina flat mount showing the
hexagonal cells of the pigmented retinal epithelium and the dendritic choroidal
melanocytes.
22 wks
K
K
d
T96
T128
T135
N85
C17
C17
PCC3/A/1
19 wks
Birth
20 wks
24 wks
11 wks
21 wks
17 wks
sc genital region
Location
1
1
1
2
1
2
3
1
1
1,2
1
1
2
1
1
1
sc base of tail
Thigh muscle
Abdominal cavity
Inside pinna
Sternum, ribs
sc head
Mid back, in muscle
Abdominal and pleural
cavity wall, in muscle
Lumbar region
in muscle
Intercostal muscle
sc base of tail
sc below eye
Attached to liver
sc below ear
sc below ear
Occipital region
cerebellum
1, 2, 3 sc neck and throat
1
no.
Tumour
Poorly differentiated
fibrosarcoma
Poorly differentiated epithelial
Cartilage
Fibrosarcoma
*
*
Sarcoma or rhabdomyosarcoma
Small, benign lymphoid
Small, fibrosarcoma
Fibrosarcoma
*
Small, cystic, necrotic
Poorly differentiated
Poorly differentiated,
some epithelium
*
Poorly differentiated
neuroectodermal
Poorly differentiated
neuroectodermal
Poorly differentiated
neuroectodermal
Nature of tumour
K = killed; d = died; sc = subcutaneous. * Not examined histologically.
19 wks
9 days
26wks
19 wks
24 wks
20 wks
K
K
d
K
T131
T136
T134
11 wks
14 wks
C17
C17
C17
C17
K
T89
C17
5 days
Birth
5 days
7 days
d
d
T72
T6
C86
C17
Birth
26 days
d
T65
C86
Birth
14 days
K
T38
C86
Birth
9 days
K
T33
C86
Birth
3 days
K
Fate
Age at
detection
of tumour
Age at
death
T18
no.
Mouse
C86
Cells
injected
Table 3. Summary of tumour bearing chimaeric mice and results of histological examination of tumours
-
—
—
—
—
+?
+
+
+
+
+
of
ECC
Presence
•»•.
vo
:s
o
O>
2
si
rs
©
>•*
Ci
n>
S3
^
**
-**
rv
^O
<^
^Si> «
osi
100
V. E. PAPAIOANNOU AND OTHERS
B
100/*
Fig. 3. Tumours from cell line C17. (A) Sarcoma or rhabdomyosarcoma from
mouse T89. (B) Fibrosarcoma from mouse T13.I.
PCC3/A/1
Seventy-one of the 86 offspring which had received PCC3/A/1 cells were
tested for chimaerism by GPI assay and examination of the coat (there is no
information on the other 15 animals). Thirty-four were test bred giving a total
of 653 progeny (average 19/mouse, range 3-47). There were four chimaeras.
One, N85, had a small lump at the base of his tail that was primarily of the
PCC3/A/1 GPI type. This animal died at 9 days of age (Table 3). Another
chimaera, N27, had a patch of agouti hairs overlying a non-chimaeric lump of
fatty tissue on her head. The other two chimaeras, both males, had small
scattered patches of agouti hairs, but no other chimaerism was evident when
these mice were assayed by GPI analysis. They had 16 and 40 progeny when
test bred but with no evidence of germ line chimaerism.
Tumours
Tumours composed largely of cell progeny of the injected cells developed
following injection of three of the four cell lines. Results of GPI analysis of the
C17 tumours are presented in Table 2 and results of examination of all tumours
are summarized in Table 3 along with the locations. There were two main
categories of tumour: those that were first detected at birth or shortly thereafter
Cultured teratocarcinoma cells in mouse embryogenesis
101
and were composed largely of undifferentiated embryonal carcinoma cells
(Fig. 1) and those that became apparent later in the animals' lives and tended to
be mainly fibrosarcomas (Fig. 3). The former, with the exception of T6, were
from cell line C86, the latter were all from cell line C17. It is interesting to note
that the early tumours were mostly encapsulated subcutaneous tumours while
those that developed later were more often located within skeletal muscle.
DISCUSSION
The fate of embryonal carcinoma cells from four in vitro cell lines has been
examined following their injection into genetically dissimilar mouse blastocysts.
These cells are capable of participation in normal embryogenesis (Papaioannou
et al. 1975) as are teratocarcinoma stem cells from in vivo tumours (Brinster,
1974; Mintz & Illmensee, 1975; Illmensee & Mintz, 1976). However, the rate
of chimaerism detected is low even after extensive GPI analysis and test
breeding, being at most 24 % for isolated C17 cells. The levels of chimaerism
were also very low, and chimaerism was sometimes detectable only by the
presence of tumours developing in the adult animals. The rate of chimaerism in
the studies using in vivo tumour cells (Mintz & Illmensee, 1975; Illmensee &
Mintz, 1976) was in the same range (16-30 %) and levels of chimaerism were
also generally low except in three cases. Germ line chimaerism was detected in
one male mouse that was predominantly derived from the injected teratoma
cells (Mintz & Illmensee, 1975). No germ line chimaeras were detected in our
study. This may be related to the fact that the level of chimaerism was typically
low. The cell lines used proved either XO or XX and would thus presumably
only be capable of forming oocytes. Test breeding in females for detection of
chimaerism is slow and in addition development of XO germ cells is likely to
be impaired (Lyon, 1974). It is also relevant that the four embryonal carcinoma
cell lines all have abnormal karyotypes as seen by G-banding in spite of normal
or near normal chromosome numbers. This may put the cells at some disadvantage in the embryo or render them incapable of forming functional
gametes.
Another factor of possible importance for the incorporation of cells into the
embryo is the difference of cell cycle times between the embryo and the injected
cells. In the embryo average cell cycle time at 6-5 days is 5 h and cycle times as
little as 2\ h are seen in a certain highly proliferative zone (Snow, 1976 and
personal communication). In contrast, the cell cycle times of the cultured
embryonal carcinoma cells range from 12 to 18 h. Unless the injected cells
shorten their cycles fairly rapidly, they are unlikely to contribute in a major
way to the developing embryo. An asynchrony of cell cycle times could also
help explain the unusual distribution of embryonal carcinoma-derived cells in
the chimaeras, most strikingly seen in the coat colour chimaeras (Papaioannou
et al. 1975). This is quite unlike the extensive contribution of the two cell types
102
V. E. PAPAIOANNOU AND OTHERS
to most tissues seen when chimaeras are formed by injection of synchronous
embryonic inner cell mass cells (Ford, Evans & Gardner, 1975). Illmensee &
Mintz (1976) have also pointed out that differences in cell adhesiveness between
ICM and injected cells may result in delayed teratoma cell integration and
account for the sporadic distribution of teratoma-derived cells in their chimaeras.
There are several possibilities to explain the high incidence of tumours in
our chimaeras. The transition that occurs when normal embryonic cells become
teratocarcinomas can be reversed as shown by the normal tissues in the chimaeras and in particular by the formation of functional sperm in one chimaera
obtained by Mintz & Illmensee (1975). This may depend on a variety of factors
such as incorporation of the EC cells into the embryo. Our experiments always
involved injection of more than one cell and it could be that the normal tissues
and the tumours were derived from different injected cells. Single EC cell
injections would clarify this point (Illmensee & Mintz, 1976).
Mintz, Illmensee & Gearhart (1975) found that the majority of dividing core
cells of OTT6050 embryoid bodies they used as an in vivo source of EC cells
for injection into blastocysts contained 40 chromosomes. However, they did
not provide a detailed karyotypic analysis of these cells. Karyotypic abnormalities have been demonstrated for all the cell lines used in the present studies,
in spite of the fact that two have a modal chromosome number of 40 (lies &
Evans, 1977; McBurney, 1976; Nicolas et al. 1976). These abnormalities could
be an important factor affecting the capacity of the cells to participate in
embryogenesis. To investigate this, injection of cultured EC cells with normal
karyotypes is now underway in our laboratory. Since all of the cell lines used
have been in culture for extended periods and are thus well adapted to culture
conditions, there may be other culture related changes, for example in cell
surface properties, genetic load or cell cycle time, that cause these cells to retain
tumour forming capacity. One explanation for the apparent rapid growth of
tumours shortly after birth would be that some injected cells behave autonomously and grow exponentially without being incorporated into the developing
embryo. The late developing tumours must be otherwise explained. Whether
they developed from injected cells which somehow remained quiescent for
several months or from their more differentiated progeny cannot be determined
from our limited information. The formation of fibrosarcomas has also been
observed following the injection into adult animals of cell lines isolated from
in vitro differentiated EC cells (J.-F. Nicolas, personal communication).
The occurrence of rapidly growing tumours may indicate a serious limitation
to the use of in vitro EC cell lines if it proves to be general. By contrast, only one
teratoma-derived tumour has been reported from the in vivo cells (Illmensee &
Mintz, 1976). However, the potential advantages of using cultured EC cells for
introduction of selected mutants into mouse strains by blastocyst injection
makes it all the more important to attempt to discover the cause of the tumours,
i.e. the reason for the incomplete recovery of normal function when these cells
Cultured teratocarcinoma cells in mouse embryogenesis
103
are returned to an embryonic environment. It is also important in order to
further define the validity of using cultured EC cells as a model of embryogenesis.
We wish to thank H. Mardon, L. Ofer, E. P. Evans, Dr C. F. Graham, Mrs M. White
and also Dr E. Ilgren who helped us with the classification of the tumours.
The work was supported by the Cancer Research Campaign, the Medical Research
Council, the Centre National de la Recherche Scientifique, the Institute National de la
Sante et de la Recherche Medicale (No. 76.4.311 AU), the National Institute of Health
(CA 16355), the Fondation pour la Recherche Medicale Francaise, and the Fondation
Andre Meyer. C.B. was supported by a short term EMBO Fellowship.
REFERENCES
R. L. (1974). The effect of cells transferred into the mouse blastocyst on subsequent development. /. exp. Med. 140, 1049-1056.
EVANS, M. J. (1972). The isolation and properties of a clonal tissue culture strain of pluripotent mouse teratoma cells. /. Embryol. exp. Morph. 28, 163-176.
FORD, C. E., EVANS, E. P. & GARDNER, R. L. (1975). Marker chromosome analysis of two
mouse chimaeras. /. Embryol. exp. Morph. 33, 447-457.
GUENET, J. L., JAKOB, H., NICOLAS, J. F. & JACOB, F. (1974). Teratocarcinome de la souris:
etude cytogenetique de cellules a potentialites multiples. Ann. Microbiol. hist. Pasteur
125, 135-150.
ILES, S. A. & EVANS, E. P. (1977). Karyotypic analysis of teratocarcinomas and embryoid
bodies of C3H mice. /. Embryol. exp. Morph. 38, 77-92.
ILLMENSEE, K. & MINTZ, B. (1976). Totipotency and normal differentiation of single teratocarcinoma cells cloned by injection into blastocysts. Proc. natn. Acad. Sci. U.S.A. 73,
549-553.
JAKOB, H., BOON, T., GAILLARD, J., NICOLAS, J. F. & JACOB, F. (1973). Teratocarcinome de
la souris. Isolement, culture et proprietes de cellules a potentialites multiples. Ann.
Microbiol. Inst. Pasteur 124B, 269-282.
KLEINSMITH, L. J. & PIERCE, G. B. (1964). Multipotentiality of single embryonal carcinoma
cells. Cancer Res. 2, 1544-1551.
LYON, M. F. (1974). Sex chromosome activity in germ cells. In Physiology and Genetics of
Reproduction, Part A (ed. E. M. Coutinho & F. Fuchs), pp. 63-71. New York: Plenum
Publishing Corporation.
MCBURNEY, M. W. (1976). Clonal lines of teratocarcinoma cells in vitro: differentiation and
cytogenetic characteristics. J. CellPhysiol. 89, 441-445.
MINTZ, B. & ILLMENSEE, K. (1975). Normal genetically mosaic mice produced from malignant teratocarcinoma cells. Proc. natn. Acad. Sci, U.S.A. 72, 3585-3589.
MINTZ, B., ILLMENSEE, K. & GEARHART, J. D. (1975). Developmental and experimental
potentialities of mouse teratocarcinoma cells from embryoid body cores. In Symposium
on Teratomas and Differentiation (ed. M. I. Sherman & D. Solter), pp. 59-82. New York:
Academic Press.
NICOLAS, J. F., AVNER, P., GAILLARD, J., GUENET, J. L., JAKOB, H. & JACOB, F. (1976).
Cell lines derived from teratocarcinomas. Cancer Res. 36, 4224-4231.
PAPAIOANNOU, V. E., MCBURNEY, M. W., GARDNER, R. L. & EVANS, M. J. (1975). Fate of
teratocarcinoma cells injected into early mouse embryos. Nature, Lond. 258, 70-73.
SNOW, M. H. L. (1976). Embryo growth during the immediate postimplantation period. In
Embryogenesis in Mammals, Ciba Foundation Symposium, pp. 53-70. Amsterdam:
Associated Scientific Publishers.
SOLTER, D., SKREB, N. & DAMJANOV, I. (1970). Extrauterine growth of mouse egg-cylinder
results in malignant teratoma. Nature, Lond. 227, 503-504.
BRINSTER,
104
V. E. P A P A I O A N N O U AND OTHERS
L. C. (1968). The development of teratomas from intratesticular grafts of tubal
mouse eggs. J. Embryo/, exp. Morph. 20, 329-341.
STEVENS, L. C. (1970). The development of transplantable teratocarcinomas from intratesticular grafts of pre- and postimplantation mouse embryos. Devi Biol. 21, 364-382.
STEVENS,
{Received 13 July 1977, revised 19 September 1977)