/ . Embryol. exp. Morph. Vol. 67, pp. 167-179, 1982
Printed in Great Britain © Company of Biologists Limited 1982
167
Formation of viable chimaeras by
aggregation between teratocarcinomas and
preimplantation mouse embryos
By C. L. STEWART 1
From the Department of Zoology, Oxford University
SUMMARY
The formation of viable teratocarcinoma-adult chimaeras, by aggregation rather than by
microinjection, is described. Aggregation chimaeras were produced using two pluripotential
EC cell lines, PSA-l/NG-2 and PSA-4/TG12. The frequency and distribution of chimaerism
were assessed, for the EC cells, in conceptuses recovered from in utero and in adults. In utero
37% of the morphologically normal conceptuses formed from PSA-l/NG-2 aggregations
and 73 % of the morphologically normal conceptuses produced from PSA-4/TG12 aggregations were found to be chimaeric. However, the frequency of chimaeric adults formed from
both cell lines was lower. The reason for this discrepancy appeared to be that in the chimaeric
conceptuses, the predominant tissues colonized by the EC cells were the extraembryonic
membranes.
INTRODUCTION
Chimaeras, which are individuals composed of two or more populations of
cells derived from different embryos, have been used to investigate a wide
variety of problems in the reproduction, genetics and development of mice (for
reviews, see McLaren, 1976; Russell, 1978). Recently, one particular class of
chimaeras has attracted much attention, namely those formed between mouse
embryos and embryonal carcinoma (EC) cells, the malignant stem cells of
teratocarcinomas (Brinster, 1974; Mintz, lllmensee & Gearhart, 1975; Mintz &
Illmensee, 1975; Papaioannou, McBurney, Gardner & Evans, 1975). Such
chimaeras have been of interest since it is hoped that EC cells may be used as
vectors for the introduction of new or mutant genes into the germ line of mice.
Furthermore, the colonization of the embryo by the EC cells has been of
interest in the study of malignancy (Pierce et al. 1979; Papaioannou, Evans,
Gardner & Graham, 1979).
Chimaeras have usually been produced using one of two techniques. Either
embryos, prior to blastocyst formation, have been aggregated together to form
a single composite embryo (Tarkowski, 1961; Mintz, 1964), or a single cell or
groups of cells have been injected into the blastocoele cavity of a blastocyst
1
Author's address: Heinrich-Pette-Institut fur Experimented Virologie und Immunologie
an der Universitat Hamburg, Martinistrasse 52, 2000 Hamburg 20, Federal Republic of
Germany.
168
C.L.STEWART
using a micromanipulator (Gardner, 1972). It is this latter method that has been
used to produce EC <-> embryo chimaeras. Here, a method is described for
producing viable EC <-> embryo chimaeras by the alternative aggregation technique and this method provides a simpler and as efficient means for introducing
EC cells into embryos. In addition to this, the colonization of tissues in the post
implantation embryo is described in detail for the first time.
MATERIALS AND METHODS
Cell lines
In this study, two cell lines were tested for their ability to form viable EC cell
chimaeras. These lines were derived from cloned cells and were PSA-l/NG-2
(obtained from Dr D. Martin) and PSA-4/TG12 (obtained from Dr M.
Hooper). The parent lines PSA-1 and PSA-4 were isolated from the same
tumour by Martin & Evans (1975 a). The tumour OTT5568 was induced by the
transfer of a 3-day 129/SvSlCP embryo to the testis of an A 2 G x 129/J ¥x male
(Stevens, 1970). Both lines were resistant to thioguanine and thus were deficient
in the activity of the enzyme hypoxanthine phosphoribosyltransferase (HPRT,
IMP: pyrophosphase phosphoribosyltransferase, EC 2.4.2.8). PSA-l/NG-2
was mutagenized with nitrosoguanidine prior to selection (Dewey, Martin,
Martin & Mintz, 1977) and PSA-4/TG12 was a spontaneous mutant (Slack,
Morgan & Hooper, 1977). Both PSA-l/NG-2 and PSA-4/TG12 (hereafter
referred to as NG-2 and TGI2) were chromosomally aneuploid, having a
trisomic chromosome no. 6, and also were XO (Cronmiller & Mintz, 1978;
Hooper, personal communication).
Maintenance in culture
NG-2 and TGI2 were routinely maintained on mitomycin treated STO feeder
cell layers (Martin & Evans, 19756) in alpha medium (Stanners, Eliceiri &
Green, 1971) without added nucleosides or deoxynucleosides, containing 10 %
(v/v) heat-inactivated foetal calf serum (FCS) (Flow Laboratories, Irvine,
Scotland) under a 10 % (v/v) CO 2 in air gas mixture. The dishes of feeder cells
were prepared as follows: 50 or 35 mm diameter plastic tissue culture dishes
(Sterilin Ltd, Richmond, Surrey, U.K.) were covered with a 1 % (w/v) gelatin
(Swine skin, type 1, Sigma Chemical Co., U.K.) solution and left at 4 °C for
30 min. Subsequently the gelatin was decanted and the dishes were covered to
confluency with STO feeder cells (Ware & Axelrad, 1972) which had previously
been treated with mitomycin C to prevent their growth (Sigma Chemical Co.,
U.K.); (technique described by Martin & Evans, 1975 &, and McBurney, 1976).
The EC cells grew as clumps on the surface of the feeder cells and the culture
medium was changed every day.
Aggregation chimaeras between embryos and teratocarcinomas 169
Genetic markers
A number of different strains of mice were used as the source for the embryos.
The strain used in each experiment depended on the particular intention of the
experiment.
Two genetic markers were used to distinguish the EC cells' progeny from
those of the host embryos. These were two allozymal variants of the glucose
phosphate isomerase Gpi-\ locus and albino or pigmentation in the coat colour
(Staats, 1980). The NG-2 and TGI2 EC lines were homozygous for the Gpi-\A
allele. Thus, the host embryos used in experiments with these lines were homozygous for the Gpi-lh allele and were either (CBA x C57BL/6) F 2 embryos
derived from (CBA x C57BL/6) F x matings or they were PO (Pathology,
Oxford) or MF1 (Olac) mice. The PO and MF1 strains were outbred and thus
individuals homozygous for the Gpi-V3 allele were selected from an initially
random-bred stock. The F 2 embryos were used in experiments where the experiment was to be concluded before birth and the PO and MF1 embryos were used
in experiments where the embryos were allowed to develop to term.
Aggregation with 8- to 16-cell stage embryos
The basic procedure was identical to that already described by Stewart (1980),
although there were changes in preparing the EC cells prior to aggregation. The
first series of experiments was performed with NG-2 EC cells. This line was
chosen, since it has the highest reported rate of chirnaerism by microinjection
(Papaioannou, 1979), and thus it provided a good test of the efficiency of
aggregation.
The clumps of EC cells were picked from the feeder layers, using a mouthcontrolled micropipette. Care was taken to minimize fibcoblast feeder cell contamination. These clumps were briefly washed in phosphate buffered saline
(PBS solution A of Dulbecco & Vogt, 1954) and then incubated for 5 min at
37 °C in 0-125 % (w/v) trypsin (Difco Ltd, U.K.) and 0-2 mM ethylene diamine
tetraacetic acid (EDTA) in PBS. Dissociation of the clumps of EC cells into
single cells or small groups was completed by gentle pipetting. Groups consisting
of 3-5 EC cells were then picked and placed into microdrops of prewarmed
tissue culture medium, maintained under paraffin oil (Boots Pure Drug Co.,
U.K.) where they were allowed to recover for 2 h at 37 °C. In later experiments,
an important modification to the method of dissociating the clumps of EC cells
was introduced. This involved incubating the clumps of EC cells in 0-5 mM
ethylene glycol bis tetraacetic acid (EGTA) in PBS for 30 min at 37 °C rather
than using EDTA-trypsin. All subsequent parts of the procedure were identical.
The host embryos were dissected from the oviducts into prewarmed Whitten's
medium (Whitten, 1971), the zona pellucidae were removed by brief exposure
of the embryos to acidified Tyrodes solution (Nicolson, Yanagimachi &
170
C. L. STEWART
#
Fig. 1. A group of PSA-l/NG-2 EC cells (arrowed) is shown sticking to two 8-cell
stage embryos.
Yanagimachi, 1975) and. then they were cultured in prewarmed, preequilibrated microdrops of Whitten's medium under paraffin oil.
For each aggregation, two embryos were placed into a single microdrop of
Whitten's medium supplemented with 10% (v/v) heat-inactivated FCS. A
single group of 3-5 EC cells was then sandwiched between the embryos, making
sure that the EC cells remained sticking to the embryos (Fig. 1). In a modification of this experiment, a single group containing the same number of EC cells
was aggregated with a single embryo. This experiment was only performed with
EGTA-dissociateH NG-2 cells.
Once all the embryos and EC cells remained sticking together, the aggregates
were cultured for 24 h, i.e. to the following afternoon ( = to the 4th day of
gestation; day 1 = day of plug). The aggregates were then transferred to the
uteri of (CBA x C57BL/6) Fx foster mothers that had been mated with vasectomized males of proven sterility. Whenever possible, controls consisting of two
embryos aggregated together were transferred to the contralateral horn of the
recipient. The aggregates were either allowed to develop to term or they were
dissected from the uteri on the 12th or 13th days of gestation. The conceptuses
recovered were dissected into the following tissues: the trophoblast, parietal
endoderm, embryo, amnion, yolk-sac mesoderm and yolk-sac endoderm. The
yolk sacs were separated into their mesoderm and endoderm components by
first rinsing in PBS and then incubating in a mixture of 0-5 % trypsin and 2-5 %
pancreatin (Difco) in calcium- and magnesium-free Tyrodes saline at pH 7-7 for
a minimum of 4 h at 4 °C (Levak-Svajger, Svajger & Skreb, 1969). Thereafter,
106
93
168
Born
NG-2<->• 2MF1 (TE)
NG-2<->- 2MF1 (EG)
T G 1 2 ^ • 2MF1 (EG)
106
85
164
56
57
201
131
37
185
17
14
25
12
10
33
18
6
27
Nos. of
recipients
6
5
6
7
6
14
6
4
10
Nos. of
pregnant
recipients
38
46
36
56
28
103
41
25
67
16
20
20
26
16
61
27
11
33
Nos. of
conceptuses
recovered
2
5
3
—
—
10
10
8
24
Nos.
chimaeric
12-5
25
15
16.4
37
73
73
/o
i
Si
s
1
5
^^
Co
s
S
»>•«.
r
(TE), EC cells dissociated by trypsin EDTA; (EG), EC cells dissociated by EGTA.
60
62
225
139
40
201
Controls
2F2
2 MF1/CFLP
Recovered from in utero
NG-2<-> 2F 2 (TE)
NG-2<-> 2F 2 (EG)
N G - 2 « 1F2 (EG)
T G 1 2 ^ 2F 2 (EG)
EC <-> embryo aggregation
Nos. of
aggregations
Nos. of
aggregations> transferred
Nos. of
embryos
transferred
to those
mice that
became
pregnant
Table 1. The number <of chimaeric conceptuses recovered from in utero and the number of chimaeric individuals born
ggrega
cinomas
172
C. L. STEWART
Table 2. The distribution of chimaerism in the conceptuses recovered from in utero.
Thefiguresin parentheses relate to the method used to prepare the EC cells before
aggregation. All aggregations were performed with 2 (C57BL/6 x CBA) F2
embryos, except where indicated
Conceptus no.; type of aggregation
NG-2—F 2 ( l )
NG-2 — F 2 ( 2 )
Embryo
••••••••••
Amnion
0000000000
Yolk-sac mesodermnnnnnnnnDn
IIIIIDDDDD
• •••OHBOO
nnannnnDin
Yoik-sac endodermnnnnnnDDDD
Parietalendoderm DDODDDDDDD
Trophoblast
OODOODDODD
DiDDDDDDii
0000000000
DODDDDOOOO
(single
NG-2—F 2 ( 2 ) embryo)
••••••••
p
OMDO
ODODDOOD
ODDOODDD
Conceptus no.; type of aggregation TG 12 —•- F 2(2)
11 13 15 17 19 21 23
Tissues analysed
1 2 3 4 5 6 7 8 9 10 12 14 16 18 20 22 24
Embryo
•••••••00000000000000000
Amnion
••DnnDDMiliDDDDCnDaDnDD
Yolk-sac mesodermBDBOODDDB • • • • • • • • • • • • • • D
Yolk-sac endodermODDDDDDDD 0 0 0 0 0 0 0 0 0 0 0 0 0 0 8
Parietal endoderm 0 0 0 0 0 0 0 0 0
Trophoblast
00000.0000
*— Not recovered - *
(1) = EC cells dissociated by trypsin - EDTA
(2) = EC cells dissociated by EGTA
_
5 uumaenc
XT-T- -
D
**
* A
H 0 S t
tyP6
NT = not tested
they were transferred to PBS at room temperature and the tissues separated
using forceps.
All of these tissues, as were the adult tissues, were screened for chimaerism
by running aqueous extracts of tissues on starch gel electrophoresis, to separate
out the allozymal GPI variants (Chapman, Whitten & Ruddle, 1971). The
technique used was sensitive enough to detect a minimum contribution of 5 %
of the minor band.
RESULTS
The ability of two EC cell lines to aggregate with 8- to 16-cell embryos and
form viable chimaeras after transfer to foster mothers has been investigated.
Only one of the lines, NG-2, has been reported to colonize embryos and produce
chimaeras (Dewey et ah 1977), whereas the other line, TGI 2, has not until now
been investigated.
The results of all the aggregation experiments are presented in Table 1. In the
preliminary series of experiments performed with trypsin-EDTA dissociated
NG-2 cells, 103 aggregates were transferred and 61 conceptuses were recovered
(only in those aggregates that became pregnant were the number of transferred
aggregates counted). Of these 61, 10 were found to be chimaeric solely in the
embryonic fraction (Table 2). None of the 61 conceptuses exhibited any colonization in the yolk sac or amnion and in none of the 37 conceptuses from which
1MF1 • NG-2 (TE)
2MF1 • NG-2 (TE)
3MF1 • NG-2 (EG)
4MF1 * NG-2 (EG)
5MF1 * NG-2 (EG)
6MF1 * NG-2 (EG)
7MF1 • NG-2 (EG)
1TG12 -> MFl (EG)
2TG12 + MFl (EG)
3TG12 + MFl (EG)
Aggregation
combination
-
+
—
—
—
+
—
—
+
_
_
+
+
-
—
+
-
-
_
_
_
_
_
_
_
_
Not tested
3
P
2-
+
r
53
Not tested (eaten)
<_
_.
§
—
w
P
C/5
-
_
_
-
-
_
_
—
—
c
—
—
_
_
+
—
+
_
_
CO
p
3
O
"d
p
—
—
_
_
3
C/3
—
—
- -
_
_
ft
3
(TE), EC cells dissociated by trypsin EDTA; (E (G,E Ccells dissociated by EGTA.
<S +
<?
+
( ? -
4wk
4wk
lOd
n
<? $
<J H
? H
<?
£
9
•o
17 wk
5 wk
3wk
27 wk
10 wk
1 wk
Iwk
Age at
autopsy
<?
3
O
a
•d
3*
ft
o
N.T. -
B*
p
i
a.
>
P
3
O
o
Table 3. Chimaeras born, shows the pattern of colonization of both EC cells in the adult chimaeras
Bl
3'
$
bo
U)
8
s-
§
I
i
Hi
5'
3
!
25
174
C. L. STEWART
Fig. 2. Mouse no. 4 produced by aggregating a group of PSA-l/NG-2 cells with
2 MFl albino embryos. Note the pigmented hair follicles derived from the embryonal carcinoma cells.
Fig. 3. Mouse 2 produced by aggregating PSA-4/TG12 cells with two MFl albino
embryos. Pigmented hair follicles, derived from the EC cells, can be seen on the
flank and head.
Aggregation chimaeras between embryos and teratocarcinomas 175
the trophoblast and parietal endoderm had also been dissected, were these
tissues colonized.
Of the 46 MFl b b <-> NG-2 aggregates that were transferred and allowed to
develop to term, 20 were born. Two were overtly chimaeric and showed extensive EC derived pigmentation in the coat. One of these also contained pigment
in the eyes and both exhibited chimaerism in some internal organs (Table 3).
No evidence for an EC derived cell population was found in the remaining 18
offspring.
These results were encouraging. However, the rate of chimaerism was less
than that reported for the microinjection experiments performed with the same
cell line (Dewey et al. 1977). Thus, a variety of methods were attempted to try
to improve the rate of chimaerism. Eventually, substitution of trypsin-EDTA by
0-5 mM EGTA in PBS was found to be the simplest and most effective (Table 1).
Disaggregation of the NG-2 cells with EGTA resulted in an increase in the
rate of chimaerism in the conceptuses (from 16 to 37 %). However, this was due
to colonization of the extraembryonic membranes, in particular the amnion
and yolk sac rather than the embryo proper. An increase in the percentage of
NG-2 chimaeras born did also occur (12-5 to 25 %) (Table 1). Similar results
were also obtained with the TGI2 EC cell line where the yolk-sac mesoderm
was colonized at the highest frequency (17 out of 24 chimaeras). In this series
of experiments, using TG12 EC cells, the rate of chimaerism was relatively high
(24 out of the 33 conceptuses recovered), although only 7 of these were chimaeric
in the embryonic fraction; a figure that was almost identical to the percentage
of live MF1 <-> TG12 chimaeras that were born (Table 1).
A series of experiments were also conducted, in which a single embryo was
aggregated with a group of 3-5 NG-2 EC cells, and although the rate of
chimaerism was high, 7 out of the 8 conceptuses that were chimaeric were
severely malformed (Table 1).
A total of 10 viable NG-2 and TGI 2 chimaeric offspring were born (Figs. 2
and 3). The majority exhibited coat colour pigmentation, with mouse no. 4
(Fig. 2) being the most extensive. Analysis of the internal tissues of the individuals revealed some to be chimaeric in many tissues and in others the chimaerism was confined to one or two organs (Table 3). No tumours were found in
any of these chimaeras, although a histological examination of the chimaeric
tissues was not undertaken. However, the presence of pigmentation in the coat
and in the eyes of some individuals, and with two individuals chimaeric in the
skeletal muscle, where the intermediate Gpi-l&b band was present indicating
fusion between myoblasts from EC cells and embryonic cells (Mintz & Baker,
1967), was evidence for the EC cells having undergone the appropriate differentiation in the tissues they colonized. Two of NG-2 chimaeras (numbers 1 and 4,
Table 3) were test mated, but neither produced any offspring. Mouse no. 1 was
autopsied at the age of 17 weeks, after he had been caged with a total of 10
females, none of which became pregnant. His testes and epididymis were
176
C. L. STEWART
examined histologically and although morphologically normal sperm were
present, many of the tubules were devoid of sperm and were packed with many
round nucleated and often necrotic cells, indicating abnormal spermatogenesis.
Mouse no. 4 was caged with seven successive males, all of known fertility. She
did not become pregnant and was autopsied at 27 weeks. Her uteri appeared to
be normal, although her ovaries were small and yellow.
DISCUSSION
The experiments described here have demonstrated that the aggregation
technique is a simple but effective alternative to microinjection, for producing
EC cell chimaeras. It is possible that the method used for introducing the EC
cells into the embryos may have had an effect on the EC cell distribution in the
chimaeras. However, a comparison between the adult NG-2 chimaeras produced
here and those produced using the microinjection technique (Dewey et ah 1977)
revealed few differences, suggesting that the technique does not influence the
distribution of the EC cells in adult chimaeras, except that in those adults
produced by aggregation, no colonization of the lungs, thymus, spleen, gut or
gonads was detected, although these tissues may be found to be chimaeric if a
larger number of chimaeras was studied. Both methods did, however, produce
individuals in which there was colonization of many tissues, and others in which
an EC cell presence was confined to a few tissues.
Little information, apart from the study of Papaioannou et al. (1979), has
been published on the pattern of EC cell colonization in conceptuses recovered
from in utero. Thus, the results presented here were the first detailed description
of the pattern of in utero chimaerism of EC cells in conceptuses. The two most
striking observations were the relatively high rate of colonization by the EC
cells, especially TG12, of extraembryonic tissues either completely derived from
the mesoderm (the yolk-sac mesoderm) or containing a high proportion of
mesodermal tissue (the amnion) (Rossant & Papaioannou, 1977). The second
striking observation was the relatively low rate of colonization of the endodermal tissues of the conceptuses. No contribution by the EC cells was found
in the parietal endoderm and only six individuals (two of them including
conceptuses with extensive malformations) were chimaeric in the visceral
yolk-sac endoderm.
The precise reasons for such a pattern are unknown, since these cells in vivo
have not mimicked the pattern of differentiation that occurs in vitro. In vitro
these cell lines, when permitted to differentiate, usually produce, at first, a
mixture of cell types with the morphological and biochemical characteristics of
visceral and parietal endoderm of the conceptus (Martin & Evans, 1975 a;
Adamson, Gaunt & Graham, 1979; reviewed Graham, 1977; and Martin,
1978). However, in vivo these cells, on the basis of the results presented here,
only colonized these tissues at a very low rate, and the reasons for this are
Aggregation chimaeras between embryos and teratocarcinomas 177
difficult to explain. Possibly, due to the EC cells' abnormal karyotype, selective
pressures exerted by the embryonic tissues restricted the EC cell differentiation
(Graham, 1977). Alternatively, the relatively low rate of colonization of the
extraembryonic endoderm could be explained on the basis of the results from
Gardner & Rossant's (1979) investigation of the inner cell mass (ICM) from
5th day blastocysts. They showed that primitive endoderm and primitive ectoderm isolated from 5th day ICM's were already committed tissues, in that they
produced two distinct distributions when reinjected into 4th day blastocysts.
The primitive endoderm exclusively colonized the extraembryonic endoderm of
the yolk sac, whereas the primitive ectoderm colonized the yolk-sac mesoderm
and embryo. Thus, it may have been possible that EC cells maintained for
prolonged periods in vitro required time to adjust to an embryonic environment,
i.e. inside the ICM, and this prevented them being exposed to the appropriate
conditions permitting endoderm formation.
Apart from again invoking the argument of selective pressures, there is no
explanation for the very high tendency, especially of the TGI2 cells, to colonize
the mesoderm in chimaeras, since there is no report of these cells predominantly
producing mesodermal cells when undergoing differentiation in vitro. The high
frequency of the TGI2 cells colonizing the yolk-sac mesoderm may have
resulted in a high proportion of the adults being chimaeric in the blood, since
it is the yolk-sac mesoderm that the first blood cells are formed (Metcalf &
Moore, 1971). However, within the haematopoietic system, cells lacking the
HGPRT enzyme are known to be selected against (Kelley & Wyngaarden, 1978)
and this may have accounted for the absence of haematopoietic cells, derived
from the TGI2 cells being present in the adults. Therefore, experiments are
being performed to discover if the wild-type parent line, PSA-4, does have a
similar tendency to colonize the yolk-sac mesoderm at high frequency and hence
the haematopoietic cells of the adult.
I would like to thank Drs C. F. Graham and V. E. Papaioannou for their advice during
the time this work was being performed. The author was supported by an M.R.C. research
studentship.
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(Received 10 April 1981, revised 20 July 1981)