/. Embryol. exp. Morph. Vol. 69, pp. 127-140,1982
127
Printed in Great Britain © Company of Biologists Limited 1982
A correlation between the
capacity of cavity formation and the subsequent
differentiation of teratocarcinoma
embryoid body lines
By KAZUKO UNO 1
From the Department of Zoology, Faculty of Science,
University of Kyoto
SUMMARY
Seven different embryoid body (EB) lines of mouse teratocarcinoma were isolated from
a single EB. With regard to each of the lines, a comparison was made of the following developmental properties, including potentiality: (1) cavity formation in a short term intraperitoneal
passage, (2) growth in vivo, (3) cardiac muscle differentiation in vitro following intraperitoneal
passage and (4) differentiation of solid tumours in vivo. These lines could be divided into
three distinct groups with respect to their capacity for cardiac muscle differentiation. It has
been shown that a high capacity for cell differentiation in vitro correlates well with the
capacity for cavity formation of an EB during the in vivo period. This cavity formation
was followed by the appearance of primitive-streak-like structures, from which mesodermal
cells were subsequently formed.
INTRODUCTION
An EB line, SEB III, isolated from" OTT 6050 of mouse teratocarcinoma is
characterized by a high capacity for cardiac muscle differentiation in vitro
following the intraperitoneal passage (Amano, Uno & Hagiwara, 1978). It
seems that the preceding in vivo passage is necessary for this type of differentiation. Thus, the question arises as to whether the EB of a particular line
represents any special characteristics in the developmental process during
in vivo passage. Several different EB lines, differing in their capacity for cardiac
muscle differentiation in vitro, have been isolated. A comparison of the in vivo
features of these lines in the course of intraperitoneal passage is expected to
show whether or not there are any specific developmental events in vivo
characteristic of these lines having a capacity for high cardiac muscle differentiation. The findings in the present paper suggest that there is a correlation
between the formation of a cystic EB in vivo (cf. Martin, Wiley & Damjanov,
1977) and the high incidence of cardiac muscle differentiation in subsequent
in vitro cultures.
1
Author's address: Department of Zoology, Faculty of Science, University of Kyoto,
Kyoto 606, Japan.
5-2
128
K. UNO
y-*z*
<0m'
Fig. 1. Morphological features of SKEB. Each bar indicates 100 microns, (a) In
vitro line consisting of dozens of embryoid bodies, many of which appear to be
small buds, (b) Histological section of in vitro embryoid bodies, showing several
cores of teratocarcinoma cells and superficial endoderm cells with a number of
large vacuoles.
MATERIALS AND METHODS
Establishment ofEB lines
EB lines were established from transplantable mouse teratocarcinoma
OTT 6050 by the methods of Amano et al. (1978). For primary cultures, a
suspension of EB was plated to 60 mm tissue culture Petri dishes in Eagle's
Minimum Essential Medium supplemented with 0-2 mM glutamine and 10%
foetal calf serum (selected batches).
To establish the EB lines, each single EB was isolated and transferred to a
new dish and cultured for several days until a large cluster of EB having dozens
of small buds was formed (Fig. 1). The clusters were dissociated by gentle
pipetting, and each EB was picked up. This procedure was repeated for more
than five times. In all steps involved in isolating a respective EB line, the
structure of the EB was maintained. The established EB lines were subcultivated
every 2 days. They were non-clonal lines but even after 3 years of continuous
culture, each EB line still had the same degree of differentiative ability. A
number of EB lines have been established from OTT 6050 according to the
procedure given above. Among these, seven different EB lines were chosen
and a comparison of several cellular and developmental features was made.
Differentiation of teratocarcinoma
129
Among these lines, SEB III was isolated from primary culture of OTT 6050
in 1975, as previously reported by Amano et al (1978). SKEB, SSEB, ASEB
and TSEB are derivatives of SEB III which were once injected into the peritoneal
cavity, and established in 1978. ODEB and OCEB were derivatives of newly
isolated primary culture of OTT 6050 in 1978.
Induction of cardiac muscle differentiation by a 7-day intraperitoneal passage
The method for intraperitoneal passage has already been described (Uno &
Amano, 1978). About 5x 103 EB of each in vitro line were injected into the
peritoneal cavity of a 129/Sv-Sl strain mouse and recovered 7 days later. An
aliquot of the suspension of the recovered EB was plated onto collagen-coated
dishes to study the ability of in vitro differentiation. The whole culture samples
were examined and the locations of pulsating foci and other differentiated
tissues were marked with a marker pencil. The frequency of cardiac muscle
differentiation was designated as the number of pulsating foci observed within
14 days of in vitro culture per number of EB plated in each culture dish.
Morphological observations of EB in the peritoneal cavity
In order to compare internal changes that occur during the intraperitofleal
passage, aliquots of the EB of seven different lines were fixed at various times
after the intraperitoneal passage with 2-5% glutaraldehyde in 0-1 M cacodylate
buffer. The recovered EB were photographed to estimate their size by measuring
the average length of major and minor axes.
The samples were then post-fixed with 1 % osmic acid in 0-1 M cacodylate
buffer, dehydrated with an ethanol series, embedded in Spurr's medium (Spurr,
1969) and spread into a single layer at the bottom of an embedding hole
(7 mm wide, 13 mm long and 2 mm deep). For observation by light microscopy, sections (3x7 mm) were serially cut at 1 [im. thick and stained with
1 % toluidine blue. The frequency of cavitation was determined by the three
photographs of randomly selected sections. The differentiation index of the
blood island was also estimated similarly.
The mitotic index was pepresented as the ratio of the number of metaphase
cells to the embryonal carcinoma (EC) cells counted (about 1000 cells). Outer
endodermal cells were excluded from the counting.
Estimation of potentiality of'm vivo differentiation
About 1000 EB were injected into the peritoneal cavity of a syngenic mouse.
After 4 weeks, the solid tumours attached to the fat body were recovered and
fixed with Bouin's fixative, embedded in paraffin, and stained with haematoxylin
and eosin. The percentage area of the histological sections occupied by each
tissue was estimated by random sampling according to the methods of Evans
(1972). A ruled eyepiece was repeatedly placed at random over the sections and
the tissue types and the areas observed were recorded. The results were calculated as the percentage of the total number of tissue types observed.
130
K. UNO
Table 1. The differentiation of pulsating foci induced by 7 days'1
intraperitoneal passage in seven different embryoid body lines
Embryoid body
lines
Frequency of
pulsating foci for each
plated embryoid body (%)
mean ± S.D.
SKEB
SSEB
SEB III
TSEB
ASEB
ODEB
OCEB
0-62 + 0-46
0-61 ±019
0-27 ±013
0008 ±0007
0003 ±0004
0
0
Fig. 2. Recovered SKEB after 1 day intraperitoneal passage. One bar indicates
100 fim. (a) Phase-contrast micrographs, {b) Histological section. Inner EC cells
are sparsely arranged and outer endoderm cells are rather smooth compared with
in vitro one.
Fig. 3. Recovered SKEB after 3 days' intraperitoneal passage. One bar indicates
100 /im. (a) Phase-contrast micrographs, (b-e) Histological sections. In (c) proliferation of EC cells are progressed. Mitotic cells ( • ) are seen. In (d) several
well-packed clusters (encircled by a dotted line) are seen. In (e) small cavities ( • )
filled with cellular debris are apparent. Macrophage-like cells (•) phagocytosized
cellular debris.
Differentiation of teratocarcinoma
131
No. of
counted embryoid
bodies
261
363
258
503
Days
in peritoneal
cavity
3
7
7
7
Embryoid body
line
SKEB
SKEB
ASEB
ODEB
35 (13-4%)
126(34-7%)
9 (3-4%)
2 (0-4%)
No. of
embryoid bodies including
cavity
3(1-1%)
21 (5-7%)
3(1-2%)
2(0-4%)
No. of
embryoid bodies including
blood island
Table 2. The number of EB including cavities and blood islands
Differentiation of teratocarcinoma
133
Fig. 4. Recovered SKEB after 7 days' intraperitoneal passage. One bar indicates
100 /xm. (a) Phase-contrast micrographs, (b) Typical population of cavitated EB.
In large EB, cavitation has progressed at several points. Arrow (-••) indicates
a blood island, (c) EB whose cavities elongated; the cells lining it have elongated
and formed columnar epithelium. In the upper EB, from the primitive streak-like
area, mesodermal cells delaminate into the space between the ectoderm and
endoderm. Typical visceral endoderm-like cells are seen, (d) Another example of
a primitive streak-like area (-v-). Mesodermal cells are delaminate into the space
between ectoderm and endoderm.
134
K. UNO
Fig. 5. Recovered ODEB after 7 days' intraperitoneal passage. Bars indicate 100 /im
each, (a) Phase contrast micrograph, (b) Histological section. The EB are all
simple in form.
RESULTS
Cardiac muscle differentiation in different EB lines
The potentiality of cardiac muscle differentiation in each of seven different
lines is shown in Table 1, and represented by the number of pulsating foci
formed in vitro culture after 7 day's intraperitoneal passage. Isolated lines
studied can be divided into three groups with respect to the incidence of cardiac
muscle differentiation (Table 1); high frequency lines, SKEB and SSEB, medium
frequency lines, ASEB and TSEB, and non-differentiative lines, ODEB and
OCEB. EB of all these lines were alike in morphology, before passage into
the peritoneal cavity.
Within two weeks of in vitro culture of SKEB, SSEB and SEB III, a variety
of cell types was differentiated in addition to cardiac muscle cells. In the
ASEB and TSEB cultures, differentiated cell types and the amount of differentiated cells were fewer than in the case of high frequency lines. In the ODEB
and OCEB cultures, only endoderm cells and EC cells appeared and rarely
fibroblastic cells were found also.
Differentiation of EB during the intraperitoneal passage
Within 1 day of intraperitoneal passage (Fig. 2), the original SKEB line
consisting of clusters of EB prior to injection, dispersed into single and simple
Differentiation of teratocarcinoma
010 -
135
SKEB
ODEB
0
3
7
Days in peritoneal cavity
Fig. 6. Mitotic indices of SKEB ( • — • ) and ODEB (O—O)
in the peritoneal cavity.
EB. Endoderm cells with a number of large vacuoles and rough surfaces
became smooth and somewhat vacuolated. Many neutrophils infiltrated these
EB in vivo.
After 2-3 days of passage (Fig. 3 a, b), mitotic figures could frequently be
observed (Fig. 3 c). Following the third day, most EB were filled with EC cells
and several well packed clusters were often formed (Fig. 3d). A cavity containing a little debris was observed in 13 % of all the specimens (Table 2, Fig. 3e).
Macrophage-like cells also infiltrated the cavity and phagocytosized the debris.
The number of macrophages increased from 3 to 7 days.
On the seventh day of the intraperitoneal passage (Fig. 4a-d), the presence
of the cavity became apparent in about 35 % of EB (Table 2). As shown in
Fig. 3d and 4b, the EC cells surrounding the cavity become elongated and
formed a tubular arrangement and developed into the columnar embryonic
ectoderm. Some large EB, whose major axes were more than 200 /im, contained
two or more cavities, while in smaller EB, only one cavity was formed for
each EB (Fig. 4b). In a few EB (7-14%) which developed into especially
expanded or spacious cavities, a third layer of cells appeared between the
ectoderm and endoderm (Wiley & Pedersen, 1977). A portion of the embryonic
ectoderm was often observed as a primitive-streak-like area from which meSodermal cells delaminated into the space between the ectoderm and endoderm
(Fig. 4 c, d). These structures are identical to the cystic EB found in earlier
SKEB
no. 1
no. 2
no. 3
ASEB
no. 1
no. 2
no. 3
ODEB
no. 1
no. 2
no. 3
Embryoid body
line
14-5
33-7
44-4
25-4
24-9
34-2
11-9
10-3
6-6
34-5
26-8
12-8
78-6
78-4
76-9
Mesenchyme
cell
14-5
4-8
1-6
Embryonal
carcinoma
cell
21
1-6
18-8
71
7-2
2-2
—
10
—
—
31
110
91
151
6-3
Muscle
1-4
4-7
103
43-6
3-6
22-2
Cartilage and
bone
23-2
241
111
14-5
31-3
190
Neural tissue
Table 3. Degree of differentiation in intraperitoneal solid tumours (%)
2-4
—
3-3
13-4
17-9
12-8
3-6
11-4
6-4
Epithelium
o
c
z
Differentiation of teratocarcinoma
137
Fig. 7. Histological section of an intraperitoneal solid tumour. Bars indicate
100 fim. (a) SKEB solid tumour consisting mainly of differentiated tissues. Neural
tissue (N), cartilage (C), epithelium (Ep) and EC cells (EC) are distinguishable.
(b) ODEB solid tumour consisting mostly of undifferentiated cells.
reports (Stevens, 1970, Pierce, Dixson & Verney, 1959). Mesoderm formation
was first observed on day 5 and the number of mesodermal cells increased on
day 7. Blood islands were found in many EB (Fig. 4 b, Table 2). Blood islands
occasionally contained many haematopoietic cells. Most of the cells in the
outer layer had a columnar or cuboidal shape typical of visceral endoderm, &nd
characteristic basally placed nuclei and extensive vacuolation (Pierce & Verrtey,
1961, Wiley & Pedersen, 1977).
As in the case of the line SKEB, dispersion into the single EB was observed
in the line ODEB after 1 day of intraperitoneal passage. After 2 or 3 days, the
EB of ODEB became distinctly different from those of SKEB. In ODEB, the
proliferation of EC cells was very slight and the cavity formation scarcely
occurred after 7 days of passage (0-4%, see Table 2). Typical and welldeveloped visceral-type endoderm was not seen (Fig. 5).
In the ASEB line, frequency of cavity formation (3-4% at 7 days) was
intermediate between the above two lines (Table 2).
138
K. UNO
Growth and mitotic activity during intraperitoneal passage
The size of SKEB increased steadily in the peritoneal cavity, while ODEB
ceased to grow after 3 days. Following this, size tended to decrease. The size
distribution of EB measured 7 days following peritoneal injection indicated
wide variation in SKEB (mean±s.D., 187 ± 123 nm), including the population
of very large EB. However, the size of ODEB became rather equalized
(118±47nm).
The mitotic index of SKEB was also very different from that of ODEB (Fig. 6).
The maximum mitotic index of SKEB was attained 2 days after injection, and
this stage corresponds to the stage of the most rapid proliferation just preceding
the compact cluster formation. Thus, the index declined, perhaps simultaneously
with the progress of the cavity formation. In contrast to this, the increase in
the mitotic index of ODEB 2 days following injection was only very slight.
The degree of differentiation in solid tumours
As shown in Table 3 and Fig. 7, a quantitative approach to the histology
of intraperitoneal solid tumours was made and compared. From 85 to 98 % of
the area of solid tumours of SKEB was composed of differentiated tissues, such
as mesenchymal cells, cartilages, bones, muscles and many kinds of epithelia.
More than 75 % of the area of the ODEB solid tumours consisted of undifferentiated stem cells, though differentiated tissues contained derivatives of
all three germ layers. Solid tumours derived from SSEB and SEB III were
similar in their appearance to those derived from SKEB. OCEB-derived tumours
were nearly the same as ODEB-derived tumours. ASEB-derived tumours were
intermediate between the tumours derived from SKEB and ODEB and about
75 % of these tumours constituted the differentiated portion. TSEB-derived
tumours were nearly the same as ASEB-derived tumours. Thus the order,
SKEB ^ SSEB ^ SEB III > TSEB ^ ASEB > OCEB ^ ODEB is similar to that
for the potentiality of cardiac muscle differentiation in vitro of these EB lines
(see p. 129).
DISCUSSION
Comparison of several developmental properties of different EB lines shows
a close correlation between the capacity of cavity formation in EB in the
intraperitoneal passage and that of subsequent differentiation both in vitro
and in vivo. EB lines of SKEB with a high capacity for cardiac muscle differentiation in vitro form considerably developed cavities enclosed by embryonic
ectoderm in the course of the passage and these lines also form highly differentiated solid tumours in vivo. But EB of ODEB lines, which scarcely form cavities
in the passage in vivo do not differentiate into cardiac muscle cells in vitro
and form only poorly differentiated solid tumours in vivo. The capacity for
differentiation of ASEB is generally between that of SKEB and ODEB. The
Differentiation of teratocarcinoma
139
degree of expression of these three developmental properties well parallels
these lines.
Although it is difficult to quantify the potency of EC cell lines, Martiri &
Evans (1975) grouped pluripotent line into two categories according to the
in vitro differentiation behaviour of various clones. When these EB are kept
in suspension culture condition, one could become cystic EB but the other
remained simple EB. They also indicated that one produced a great proportion
of mature tissues and relatively less EC cells than the other (Evans & Martin,
1975). Earlier works also suggest that better differentiating clones generally
contain both larger and more frequent instances of tissue types, the differentiation index roughly reflecting the total volume of differentiated tissue within
the tumours (Kahan & Ephrussi, 1970, Rosental, Wishnow & Sato, 1970).
From the present observations several steps in the differentiation of SKEB
in vivo can be identified: (1) proliferation of EC cells, (2) the formation of
well-packed clusters, (3) the cavity formation, (4) the development of embryonic
ectoderm and (5) the mesoderm formation from the primitive-streak-like area
between ectoderm and endoderm. The pattern of development observed under
these conditions resembles the development of the 5-day mouse embryo into
an 8-day one (Bonnevie, 1950). Using a particular cell line which develops into
complex and cystic EB in a suspension culture, Martin et al. (1977) demonstrated that the process of differentiation in vitro resembles the early development of mouse embryo up to the stage of mesoderm formation. In lung
colonies, three steps were identified (Ishikawa & Hagiwara, 1977; Ishikawa,
1979). From these results, it is confirmed that the process of differentiation
from EB is very similar to the normal development of mouse embryos.
From the present observations on the differentiation of SKEB and ODEB
lines, it is suggested that the cavity formation is a crucial step for subsequent
differentiation of EC cells. It is possible that the cavity formation may provide
the two-dimensional surface for the positional organization necessary for
further cell determination.
The process of myocardiogenesis from EC cells is obscure. It is known that
the heart starts to beat very shortly after the primitive streak stage (Manasek,
1978). It is possible that EB develops up to the stage of mesoderm formation
or cavity formation during the period preceding the in vivo passage. Subsequently,
cardiac muscle differentiation and some other differentiations progress in
vitro.
During the intraperitoneal periods, several kinds of ascites cells originating
from host mouse were observed in EB. But the correlation between these cells
and the development of EB is not clear as yet.
The author wishes to express her appreciation to Drs T. S. Okada and M. Yoneda for their
encouragement and critical reading of this manuscript, and to Drs S. Amano and T. Ishikawa
for their valuable comments.
140
K. UNO
REFERENCES
AMANO, S., UNO, K. & HAGIWARA, A. (1978). Cardiac muscle differentiation in vitro from
a characteristic cell line isolated from mouse teratocarcinoma. Devi Growth Different. 20,
41-47.
BONNEVIE, K. (1950). New facts on mesoderm formation and proamnion derivatives in
the normal mouse embryo. /. Morph. 86, 495-545.
EVANS, M. J. (1972). The isolation and properties of a clonal tissue culture strain of pluripotent mouse teratocarcinoma cells. /. Embryol. exp. Morph. 28, 163-173.
EVANS, M. J. & MARTIN, G. R. (1975). The differentiation of clonal teratocarcinoma cell
cultures in vitro. In Roche Symposium on Teratomas and Differentiation (ed. M. Sherman
& D. Solter), pp. 237-250. New York: Academic Press.
ISHIKAWA, T. & HAGIWARA, A. (1977). The process of differentiation of embryoid bodies
in the lung of syngenic mice. Devi Growth Different. 19, 329-335.
ISHIKAWA, T. (1979). Growth and mitotic activity of lung colonies derived from mouse
teratocarcinomas. Devi Growth Different. 21, 169-174.
KAHAN, B. W. & EPHRUSSI, B. (1970). Developmental potentialities of clonal in vitro
cultures of mouse testicular teratoma. J. natn. Cancer Inst. 44, 1015-1036.
MANASEK, F. J. (1978). Heart development: interactions involved in cardiac morphogenesis.
In The Cell Surface, vol i (ed. G. Poste & G. L. Nicolson), pp. 377-419. North-Holland.
MARTIN, G. R. & EVANS, M. J. (1975). The formation of embryoid bodies in vitro by
homogeneous embryonal carcinoma cell cultures derived from isolated single cells. In
• Roche Symposium on Teratomas and Differentiation (ed. M. Sherman & D. Solter), pp.
167-187. New York: Academic Press.
MARTIN, G. R., WILEY, L. M. & DAMJANOV, I. (1977). The development of cystic embryoid
bodies in vitro from clonal teratocarcinoma stem cells. Devi Biol. 61, 230-244.
PIERCE, G., DIXSON, F. & VERNEY, E. (1959). Testicular teratomas. I. Demonstration of
teratogenesis by metamorphogenesis of multipotential cells. Cancer 12, 573-583.
PIERCE, G. B. & VERNEY, E. L. (1961). An in vitro and in vivo study of differentiation in
teratocarcinoma. Cancer 14, 1017-1029.
ROSENTAL, M. D., WISHNOW, R. M. & SATO, G. H. (1970). In vitro growth and differentiation
of clonal populations of multipotential mouse cells derived from a transplantable testicular
teratoma. J. natn. Cancer Inst. 44, 1001-1041.
SPURR, A. R. (1969). Low-viscosity expoxy resin embedding medium for electron microscopy. /. Ultrastruct. Res. 26, 31-43.
STEVENS, L. C. (1970). The development of transplantable teratocarcinoma from intratesticular graft of pre- and postimplantation mouse embryos. Devi Biol. 21, 364-382.
UNO, K. & AMANO, S. (1978). Introduction of myocardiogenesis from mouse teratocarcinoma.
Devi Growth Different. 20, 269-273.
WILEY, L. M. & PEDERSEN, R. A. (1977). Morphology of mouse egg cylinder development
in vitro: A light and electron microscopic study. J. exp. Zool. 200, 369-402.
(Received 8 July 1981, revised 6 January 1982)