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The development of cystic embryoid bodies in vitro from clonal teratocarcinoma stem cells
DEVELOPMENTAL
BIOLOGY
61, 230-244 (197’i’l
The Development
of Cystic Embryoid Bodies in Vitro from Clonal Teratocarcinoma Stem Cells R. MARTIN’
GAIL Department
of Anatomy
and Cancer Research Institute, University 94143
of Radiobiology,
University
San Franciscd,
California
M. WILEY~
LYNN Laboratory
of California,
of California,
San Francisco,
California
94143
AND IVAN Department
of Pathology, Received April
University
DAMJANOV~ of Connecticut,
Farmington,
Connecticut
06032
20, 1977; accepted in revised form July 27, 1977
Certain clonal teratocarcinoma stem cell lines can be maintained in the undifferentiated state or can be stimulated to differentiate by a simple alteration in the culture conditions. In these cultures, differentiation occurs in a clearly definable, relatively synchronous sequence of events, as the entire cell population participates in the process of “cystic embryoid body” formation. We have studied the morphological changes that occur during this process. The pattern of development in the embryoid bodies is remarkably similar to that in normal mouse embryogenesis up to the time of formation of the third germ layer, the mesoderm. Whereas, in some embryoid bodies, mesoderm appears to form in a relatively normal manner, in moat it apparently arises by a process which has not previously been described. These results are discussed in comparison with similar studies on the development of isolated mouse inner cell masses in vitro. INTRODUCTION
The close similarity between mouse embryonal carcinoma cells, which are the stem cells of the tumors known as teratocarcinomas, and the cells of the early embryo has been dramatically demonstrated by the results of Mintz and Illmensee (1975; Illmensee and Mintz, 1976). In their experiments, embryonal carcinoma cells injected into mouse blastocysts were ’ Send reprint requests to Gail Martin, Department of Anatomy, University of California, San Francisco, California 94143. * Present address: Department of Anatomy, University of Virginia, Charlottesville, Virginia 22901. 3 Present address: Hanemann Medical College, 230 N. Broad Street, Philadelphia, Pennsylvania 19103.
found to participate with the host blastocyst cells in the formation of normal chimerit animals, which were born after the operated embryos were transferred to foster mothers. Not only were single embryonal carcinoma cells found to form a variety of somatic tissues, but also the tumor stem cells formed functional sperm in the chimeric animals. Thus, like the cells of the early embryo, embryonal carcinoma cells are totipotent. That is, they are capable of multiplying in the undifferentiated state and of giving rise to all of the differentiated cell types in an animal. The “embryonic” nature of embryonal carcinoma cells has also been demonstrated in other kinds of experiments. For example, when a solid teratocarcinoma is 230
Copyright 0 1977by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN
0012-1606
MARTIN,
WILEY,
AND DAMJANOV
injected into the peritoneal cavity of an adult mouse, the pluripotent stem cells can form embryonic structures known as “embryoid bodies” (Stevens, 1959; Pierce et al., 1959). These bear a superficial resemblance to mouse blastocysts of approximately 3.5days gestation, and are sometimes mistaken for them. However, in the blastocyst, the outer cell layer is trophectoderm surrounding an inner cell mass (ICM), while the embryoid bodies have an outer layer of endoderm surrounding an embryonal carcinoma core and do not contain any trophoblast derivatives. Such embryoid bodies are therefore more directly comparable to the inner cell mass derivatives of a normal embryo of approximately 5-days gestation, when the ICM has developed an outer layer of endoderm. These two-layered teratocarcinoma derivatives, known as “simple” embryoid bodies, will sometimes develop in the peritoneal cavity into more complex “cystic” embryoid bodies, which have numerous similarities to older embryos. It has also been shown that simple embryoid bodies removed from the peritoneal cavity can differentiate into cystic embryoid bodies in vitro and that this process parallels normal embryonic development (Pierce and Verney, 1961; Hsu and Baskar, 1974). The advantages of using the tumor stem cells as a substitute for the cells of the normal early embryo in many kinds of experimental studies have long been recognized. However, the usefulness of transplantable tumor lines is limited by the heterogeneity of the cell population and by the exposure of the cells to indeterminate influences in the host environment. Both of these limitations have been overcome since undifferentiated teratocarcinoma stem cells have been cloned and cultured in vitro (reviewed by Damjanov and Solter, 1974; Martin, 1975). The potential usefulness of such pluripotent cell lines, however, is to a certain degree dependent on their ability to differentiate in vitro in a manner that parallels normal
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embryogenesis. Most of the embryonal carcinoma cell lines that are available have not been reported to mimic normal embryonic morphogenesis to any significant extent in the course of their differentiation, although some of these cell lines can form derivatives of all three germ layers after several weeks of culture under the appropriate conditions (see review by Graham, 1977). In contrast, the clonal embryonal carcinoma cell cultures isolated by Martin and Evans (1975a,b) differentiate in a specific sequence of events, as the entire population participates in the relatively synchronous formation of embryoid bodies. These embryonal carcinoma cells are maintained in the undifferentiated state by frequent subculture in the presence of fibroblastic “feeder” cells: When passaged in the absence of feeder cells, the embryonal carcinoma cells form rounded aggregates, and differentiation begins with the formation of a layer of endodermal cells over the whole outer surface of these aggregates. Some lines of these clonal embryonal carcinoma cells stop their differentiation at this stage and remain as two-layered, simple embryoid bodies when kept in suspension; However, Martin and Evans (1975a,b) have noted that, under the same conditions, other clonal teratocarcinoma stem cell lines will continue to differentiate to complex, cystic embryoid bodies. Since such clonal cell cultures are being used as an in vitro model system for studying various aspects of early embryonic development, such as X-chromosome inactivation (Martin et al ., submitted for publication), the primary objective of the present study was to analyze in some detail the process of cystic embryoid body formation by these teratocarcinoma stem cell lines. In so doing, we hoped to determine the extent to which these cells, like the transplantable tumor lines passaged in ho, differentiate in a pattern that resembles the early postimplantation development of the mouse embryo. In making the
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comparison between embryoid body formation and normal embryogenesis, we have paid particular attention to the development in vitro of isolated mouse ICMs, as described by Wiley et al. (submitted for publication); such a comparison is highly relevant, since both the embryoid bodies and the isolated ICMs begin their development with the formation of an outer layer of endoderm, and, unlike the situation in the intact embryo, differentiation continues in the absence of trophoblastic tissue. MATERIALS
AND
METHODS
All clonal embryonal carcinoma cell lines were derived from isolated single cells as previously described (Martin and Evans, 1975a, b). Unless otherwise noted, the culture medium used in these experiments was Dulbecco’s modified Eagle’s medium (containing 4.5 g/liter of glucose) supplemented with 10% calf serum (Gibco, selected batches). Cultures were incubated in 5% CO* (in air) at 37°C. The clonal cell line PSAl forms embryoid bodies that become cystic when kept in suspension. It was isolated from secondary cultures of transplantable tumor OTT 5568, which was initiated by the transfer of a 3-day mouse embryo to an ectopic site (Stevens, 19701. The cell line SCC-S2 forms embryoid bodies that remain simple when kept in suspension. It is a clonal derivative of the main stock of the SIKR teratocarcinoma cell line, also derived from tumor OTT 5568, which has been previously described (Evans, 1972; Martin and Evans, 1974, 1975b). Stock cultures of the two pluripotent embryonal carcinoma cell lines were maintained in the undifferentiated state by subculturing the cells every 3 days and seeding them on a confluent feeder layer of Mitomycin C-treated ST0 mouse cells (Martin and Evans, 1975a). To induce embryoid body formation, the cells were seeded at lo7 cells per g-cm tissue culture dish. On the third day after
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this passage, in the absence of feeder cells, clumps of cells had formed. These were then detached from the dish by gently pipetting fresh medium over them. The detached clumps were allowed to settle for several minutes, washed in fresh medium, and then seeded in a bacteriological petri dish. The medium was changed daily during the subsequent 11-day culture period of the cells in suspension. At the time of detachment, and at 12- or 24-hr intervals thereafter, an aliquot of several hundred cell clumps was fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, and subsequently postfixed with 2% 0~0, in the same buffer. The cells were dehydrated in a graded series of ethanol in water, during which they were stained en bloc with uranyl acetate. After complete dehydration by passage through propylene oxide the clumps were embedded in Epon resin and sectioned for either light or transmission electron microscopy, as previously described (Wiley and Pedersen, 1977). RESULTS
Cystic Embryoid Body Formation Observed in the Phase-Contrast Microscope
as
In order to initiate cystic embryoid body formation, PSAl cells were plated on tissue culture dishes in the absence of feeder cells, at a time designated as Day 0 (D,). After 3 days of culture (D3), the cell clumps which had formed were detached and plated (at a time designated as D, + J in bacteriological petri dishes to which they do not adhere. The changes that occurred in the clumps over an 11-day culture period in suspension (D3 + 1, D, + 2, etc.) were followed by phase-contrast microscopy. At the time of detachment, the rounded PSAl embryonal carcinoma cell clumps appeared to be homogeneous with respect to cell type and had a smooth, refractile surface (Fig. 1A). A small fraction of the
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FIG. 1. Cystic embryoid body formation, as observed in the phase-contrast microscope. (A) Clumps of embryonal carcinoma cells at the time of detachment from the tissue culture substratum (D, + ,J. Approx. 200 x . (B) After 2 days in suspension CD, + 2) the embryonal carcinoma cell clumps have become simple embryoid bodies. Pointers indicate the outer layer of endoderm. Approx. 200 X. (0 By D, + B a cavity is apparent in most of the embryoid bodies. Approx. 200 X. (D) A large proportion of the embryoid bodies develops balloon-like cysts by Day 10 in suspension. Approx. 90 x .
clumps, however, did have some obvious endodermal cells on their outer surface. Within 24 hr of detachment (II3 + J, all of the clumps had an outer layer of endodermal cells surrounding an inner cell core and had thus become simple embryoid bodies. There was an apparent increase in clump size, probably due both to clump aggregation and to cell multiplication. By 48 hr after detachment (II3 + 2), the endodermal cell layer of these embryoid bodies had become more clearly delineated (Fig. 1B); no other changes were apparent.
At 4 days after detachment (OS + J most of the embryoid bodies were still composed of a solid core surrounded by a layer of endoderm, although in a small fraction of the population a cavity was apparent. By 5 to 6 days after detachment, most of the embryoid bodies showed internal changes: The cells had become arranged around what appeared to be an eccentrically placed cavity (Fig. 1C). After 6 days in suspension, some of the embryoid bodies began to expand and to show a balloonlike swelling on one side. Over the next
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few days, a large proportion of the embryoid bodies developed similar balloonlike cysts; these structures continued to enlarge and often reached several millimeters in diameter (Fig. 1D). Although the more solid portion of the embryoid bodies usually remained attached to the balloon-like cyst, it often appeared to atrophy, and in some cases to disappear altogether. Structural Analysis of the Process of Cystic Embryoid Body Formation In order to compare the internal changes that occurred during the process of cystic embryoid body formation with those that occur during development of the early embryo, aliquots of the embryoid bodies were fixed at various times after detachment, embedded, and sectioned. Approximately 50 embryoid bodies were examined at each time point. Although the morphological features described here characterized the embryoid bodies as a population, it should be noted that the progression of the population through each successive stage in development was not perfectly synchronous. (A) Clump structure at the time of detachment. Light-microscopic examination of the cell clumps fixed at the time of detachment indicated that they were not as morphologically homogeneous as they had appeared in the phase-contrast microscope. Although some clumps did consist of only embryonal carcinoma cells, in most there were one or more endodermal cells on the surface of the clump. In a few cases, a crescent of endodermal cells was present (Fig. 2). However, in none of the clumps fixed at the time of detachment did there appear to be a complete layer of endodermal cells encircling the embryonal carcinoma cells. Similar results were obtained when clumps were detached at an earlier time (i.e., D, + ,,, 2 instead of 3 days after plating). The structure of the endodermal cells that were present at the
FIG. 2. Clump of embryonal carcinoma cells shortly after detachment from the substratum. Pointers indicate a crescent of endodermal cells. Approx. 400 x.
time of detachment of the clumps is described below [Section (B)]. Our observations on the fine structure of the embryonal carcinoma cells correspond with previously published descriptions of such cells (Pierce and Beals, 1964; Pierce et al., 1967; Damjanov et al., 1971). They had large, irregularly shaped nuclei that contained two or three large nucleoli (Fig. 3A). Euchromatin predominated over heterochromatin, although numerous dense bodies were scattered throughout the nucleoplasm. The cytoplasm contained many free ribosomes, a few small spherical mitochondria, occasional profiles of nondistended rough endoplasmic reticulum, moderately developed Golgi, and a few membrane-bound dense bodies. We found that intracisternal A particles were not readily apparent. At the time of detachment, the embryonal carcinoma cells were closely packed and there were occasional intercellular clefts (Fig. 3A). Although a few cell junctions were present, they looked rather primitive and resembled zonulae adherents. No intercellular material was found between the cells. Those embryonal carcinoma cells on the outer surface of the clumps were mostly cuboidal, with ran-
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FIG. 3. Fine structural details of the cells present at the early stages of embryoid body formation in uitro. 4105 x (A) Undifferentiated embryonal carcinoma cells. The nucleus (N) contains prominent nuclei (n) and numerous dense bodies. (B) Cuboidal cell at the surface of a clump at D3 + 1. This cell contains profiles of rough endoplasmic reticulum (rer) distended with a flocculent material, and is joined to its neighbor by apical cell junctions (arrow). (C) Parietal endoderm cell. The cytoplasm contains distended profiles of rer, as well as some extracellular material that resembles Reichert’s membrane km) along its basal aspect. (D) Visceral endoderm cell. Many apical microvilli and electron-lucent vacuoles (VI distinguish this cell type from parietal endoderm cells and from embryonal core cells.
domly distributed organelles and no apical microvilli or vacuoles. (B) Morphological features of endodermal cells. The endodermal cells of embryoid bodies were of two distinct types, corresponding to the two types of primary endoderm found in the mouse embryo. One resembled parietal (distal) endoderm, as distinguished by its distended profiles of rough endoplasmic reticulum (RER; Figs. 3B and C). Within these dilated RER, and also as a thick layer underlying these parietal endodermal cells, was a substance thought to be analogous to the mucoprotein Reichert’s membrane of the mouse embryo. The second cell type re-
sembled visceral (proximal) endoderm, as identified by its narrow profiles of RER, numerous apical electron-lucent vacuoles, and numerous microvilli (Fig. 3D). Reichert’s membrane-like material was also occasionally observed underlying these visceral endodermal cells. Both types of endoderm were present in roughly equal proportions, although one or the other type often predominated on an individual embryoid body. The distinction between the two cell types, however, was not always clear-cut, and occasional, intermediate forms were observed. During the 24-36 hr that followed detachment of the clumps, the endodermal
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cells formed a continuous layer around the clumps. Endoderm formation appeared to begin with the flattening of outer cells, which was accompanied by the formation of apical junctions and an increase in dilated RER, so that the cells resembled parietal endoderm (Figs. 3B and 0. Shortly after the appearance of these features, cells that had the characteristics of visceral endoderm were also apparent in the outer cell layer. From our observations, it is not possible to say whether these arose from cells initially resembling parietal endoderm or whether they arose directly from the embryonal carcinoma
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cells. Interestingly, intracisternal A particles were common in these visceral endoderm cells, but were not apparent in parietal endoderm. (C) Cavitation and formation of a columnar epithelium. About 4 to 5 days after detachment, internal changes in the cores of the embryoid bodies became apparent. The first change was the appearance of one or more small cavities full of cellular debris (Fig. 4A). These usually formed two to three cell diameters from the outer surface; since the embryoid bodies were often more than 10 cells in diameter, the cavity therefore formed eccentrically, thus
FIG. 4. Cavitation and formation of a columnar epithelium in embryoid bodies. (A) First appearance of a cavity at approximately D, + ,. Two or three cell diameters from the outer endodermal cell layer (en), a small cavity (cav) filled with cellular debris becomes apparent. Approx. 400 X. (B) Detail of core cells surrounding debris-filled cavity shown in Fig. 4A. These cells are joined by apical cell junctions (arrows). 4105 X. (Cl Shortly after its initial appearance, the cavity was enlarged and the cells lining it have elongated and formed a columnar epithelium. Approx. 400 x. (D) Typical population of cavitated embryoid bodies at D, + ,. Approx. 100 x.
MARTIN, WILEY, AND DAMJANOV
establishing polarity in the embryoid bodies. The embryonal carcinoma cells surrounding this cavity had formed junctional complexes bordering the cavity (Fig. 4B), while embryonal carcinoma cells elsewhere in the cores of the embryoid bodies were no different from the undifferentiated cells at the time of detachment and showing only unspecialized junctions between them. At the time that the cavity formed, or shortly thereafter, the embryonal carcinoma cells that surrounded the cavity elongated to create a simple epithelium of columnar cells (Fig. 40. This process is analogous to the formation of the proamniotic cavity and embryonic ectoderm in the normal mouse embryo. These changes were relatively synchronous in the population as a whole, and all of the embryoid bodies developed cavities enclosed by a layer of columnar cells (Fig. 4D). The apparent heterogeneity of the population in Fig. 4D is probably a consequence of variation in the plane of section through these asymmetric embryoid bodies. (0) Mesoderm formation in cystic embqoid bodies. In the normal development of the embryo, mesoderm differentiation follows the formation of the proamniotic cavity and columnar ectodermal epithelium: A primitive streak, or thickening, on one side of the embryonic ectoderm is established and mesodermal cells then delaminate into the space between the ectoderm and the endoderm, thus forming a third layer of cells (see Snell and Stevens, 1966). Evidently, mesoderm could form in an analogous way in the embryoid bodies, since we found at least two clear examples of this (out of approximately 200 embryoid bodies examined at this stage), as shown in Fig. 5. In these embryoid bodies, a third layer of loosely arranged cells was found between the endoderm and the ectoderm. Observations of these cells in the transmission electron microscope indicated that they did not form junctional complexes with one another (Fig. 5B), in
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contrast to the cells in both the endodermal and ectodermal cell layers, which usually did. The cells in this third layer resembled mesoderm, in that they were oriented lengthwise in the cleft between the endoderm and ectoderm, lying outside of any basal lamina that was present under the ectoderm or endoderm. Also, like mesodermal cells in intact cultured embryos (Wiley and Pedersen, 19771, they contained fusiform nuclei and had many free ribosomes, but few that were membrane bound. Although mesoderm formation apparently occurred in most of the embryoid bodies, it did not necessarily arise by a process analogous to normal development. Except for the two examples described above, we found little evidence of mesoderm delamination. Between D, + 6 and D 3 + 109 many embryoid bodies underwent expansion of the cavity and attenuation of their columnar epithelial cells (Fig. 6A). The cells lining the cavity thus came to resemble the mesodermal cells which were subsequently observed in the cultures (see below). Many of the balloon-like structures which resulted from this cavity expansion and cell attenuation retained a focus of morphologically undifferentiated embryonal carcinoma cells at one pole (Fig. 6B). (At first glance, this particular example looks misleadingly like a mouse blastocyst, but since the wall of the cyst is two cell layers thick, the outer one of which is visceral endoderm, this structure is in no way analogous to a blastocyst.) In some embryoid bodies, the cells underlying the endoderm appeared to indent into the cavity (Fig. 60. By lo-11 days in suspension, the population of embryoid bodies had become considerably more pleiomorphic and was found to contain complex double cysts of the type shown in Fig. 7, in addition to the single cysts described above. Our observations did not provide any clear indication of how the double cysts might have arisen from the less-complex structures.
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FIG. 5. Mesoderm formation in embryoid bodies. (A) Between the outer endoderm (en) and the inner ectodermal layer fee) is a third layer of fusiform cells which resemble mesoderm (mes). Approx. 400 x (B) Ultrastructure of the embryoid body shown in Fig. 6A. A mesodermal cell (mea) occupies the cleft between the endoderm (en) and ectoderm (ec). 5941 x. (C) Another example of a primitive streak-like area. Approx. 400 x.
Such double cysts were enclosed by one continuous layer of endoderm and contained two inner vesicles, one of which was expanded and attenuated, while the other resembled the unexpanded columnar ectodermal vesicle present earlier in the culture period (Figs. 7A and B). In some cases, however, this unexpanded vesicle appeared to have atrophied (Figs. 7C and D) or even disappeared altogether. Structures which resembled blood-filled capillaries were often observed in the attenuated cell layer lining the balloon-like cyst walls (Fig. 7E), thus suggesting that
the mesoderm lining the cyst walls arose from the attenuated cells. These balloonlike cysts were remarkably similar to the yolk sac which develops in the embryo between 7 and 8 days of gestation. In none of the embryoid bodies that we examined did we observe any cells which resembled extraembryonic ectoderm or its derivatives. This is not surprising, since the extraembryonic ectoderm is thought to be a trophoblast derivative (Gardner and Papaioannou, 1975) and there is no evidence that embryoid bodies ever contain any trophoblastic cells. We also did
MARTIN,
WILEY,
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FIG. 6. Ectoderm attenuation in cyst and apparently attenuated inner attenuated ectoderm and a focus of indentation of ectoderm (ie). Approx.
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expanding cystic embryoid bodies. (A) Embryoid body with a single layer of ectoderm at D, + 1,,. Approx. 400 x. (B) Embryoid body with undifferentiated cells (u). Approx. 150 X. (C) Embryoid body with 150 x
not observe any clear examples of “paired cysts” of the type frequently formed by isolated ICMs cultured in vitro (Wiley et Neither al., submitted for publication). did we observe the “budding” leading to polycyst formation which occurred in the cultures of embryoid bodies removed from the peritoneal cavity (Hsu and Baskar, 1974). Studies of Teratocarcinoma Stem Cells that Form Only Simple Embryoid Bodies As noted above, certain clonal embryonal carcinoma cell lines form embryoid bodies which remain simple when kept in suspension (Martin and Evans, 1975a, b). These cells, like those that form cystic embryoid bodies, can, however, differentiate to a variety of cell types such as
cartilage and keratinizing epithelium if the embryoid bodies are allowed to attach to a substratum (Martin and Evans, 1975c; Evans and Martin, 1975). We have compared the behavior in suspension culture of one such cell line, SCC-S2, with that of the PSAl cells that form cystic embryoid bodies, in the hope of gaining some insight into the cause of the differences in their differentiative patterns. In the undifferentiated state, there were no apparent morphological differences between the two cell types. Under conditions that allow development into embryoid bodies, however, we observed one striking difference between them. In contrast to the cystic embryoid bodies, which had a mixture of both visceral and parietal endoderm, virtually all of the endoderm on
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FIG. 7. Features of balloon-like cysts present in the embryoid body population by D, + ,O. (A) Detail of ectodermal vesicle (ec) present in a double cyst. Note the single layer of endodermal cells (en) surrounding both cavities and the single attenuated cell layer lining the larger cavity. Approx. 400 x. (B) Low magnification of the double cyst shown in Fig. 7A. Approx. 100 X. (C) Another double cyst, with a somewhat atrophied ectodermal vesicle (ec). Approx. 100 X. (D) Detail of the ectodermal vesicle of the double cyst shown in Fig. 7C. Approx. 300 x (El Typical capillary-like structure found in the attenuated cell layer of the balloon walls. Approx. 300 x.
the simple embryoid bodies formed by the SCC-S2 cells was of the parietal type. This probably explains the observation that the cores of these embryoid bodies rapidly became encased in a very thick layer of Reichert’s membrane-like material and thus after several days in suspension the endodermal cells often detach from the outer surface. We examined approximately
200 simple embryoid bodies at various times in the course of the experiment, and on only one occasion did we observe a small number of outer cells of SCC-S2 embryoid bodies that was clearly of the visceral type. This difference in the type of endoderm formed probably accounts for the earlier observation (Martin and Evans, 1975c) that, when simple and cystic em-
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bryoid bodies attached to a substratum, the endodermal cells which migrated away from the cores of these two types were morphologically different from one another. DISCUSSION
The observations described here demonstrate the similarity in the fundamental pattern of development of cystic embryoid bodies formed in vitro from clonal teratocarcinoma stem cells and of the ICM of the mouse embryo; in both cases, the first differentiative event is the formation of an outer endodermal cell layer. This is followed by cavitation and subsequent formation of a columnar epithelium by the inner cells. At the stage of mesoderm formation, however, there is an apparent deviation from the normal pattern of development in the embryoid bodies. Although mesoderm can form as a third layer between the endoderm and ectoderm, in most cases it appears to arise by some other process. That this abnormal method of mesoderm formation is not an anomaly intrinsic to teratocarcinoma cells, however, has been clearly demonstrated by the work of Wiley et al. (submitted for publication), which showed that, in the large majority of isolated inner cell masses cultured in vitro, mesoderm also apparently develops by some method other than formation of a third layer of cells. This latter observation underscores the need for comparing the development of embryoid bodies with that of the isolated ICM in vitro, rather than with the behavior of the intact embryo. In both the embryoid bodies and the isolated ICM cultured in vitro, development proceeds independently of trophoblast-derived tissues, although one cannot rule out the possibility that contact with the trophoblast prior to isolation of the ICM might have some determinative effects on the cells of the ICM. Such a comparison is shown in Table
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1, which is based on our observations and those of Wiley et al. While the similarities between the development of cystic embryoid bodies and isolated ICMs cultured in vitro are striking, there are also differences. One of these concerns the type of endodermal cells present. In the normal embryonic egg cylinder in utero and also in the experiments of Wiley et al ., the endoderm covering the ICM and its derivatives is the visceral type only. Thus, a thick layer of Reichert’s membrane, a parietal endoderm product, is not observed between the embryonic ectodermal epithelium and the endoderm. In contrast, in our cultures, the cystic embryoid bodies always contained a high proportion of parietal endoderm, as evidenced by the thick layer of Reichert’s membrane-like material between the inner columnar epithelium and the outer endodermal cell layer, shown in Fig. 4C. Experiments designed to determine if differences in the culture medium employed in this and the study by Wiley et al. were responsible for the type of endoderm formed indicated that this was not the case. However, cells of the parietal type have been reported to form part of the endodermal layer covering ICM derivatives in both intact embryonic egg cylinders cultured in vitro (Solter et al. 1974) and isolated inner cell masses cultured in vitro (Strickland et al., 1976; Solter, personal communication). At present, there is no explanation of why in some cases embryonic material cultured in vitro apparently produces a mixture of parietal and visceral endoderm and in other cases it does not. However, the fact that cells with features of both types of endoderm can be found in our cultures suggests that the two endodermal cell types may be closely related. This idea is supported by the observation of Diwan and Stevens (1976) that isolated visceral endoderm grafted to the testes of adult mice can produce large amounts of Reichert’s membrane.
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COMPARISON OF TERATOCARCINOMA CYSTIC EMBRYOID BODY FORMATION WITH MOUSE INNER CELL MASS DEVELOPMENT in Vitro Stage of development Endoderm
Cavitation columnar
in vitro
formation
and formation epithelium
of a
Features of teratocarcinoma tic embryoid bodies
cys-
formation
of isolated
ICMs”
Endodermal cell layer is a mixture of both visceral and parietal types. Readily attach to a tissue culture substratum at all stages of development.
Endodermal cell layer is almost exclusively of the visceral type. b Little or no attachment to a tissue culture substratum after endoderm formation.
Cavity
Cavity forms centrally and all of the inner cells form a columnar epithelium surrounding the cavity.
formation
may be eccentric.
Columnar epithelium forms around the cavity, but, in cases where the cavity is eccentric, the columnar epithelium does not completely surround the cavity and some cells apparently remain undifferentiated. Mesoderm
Features
Mesoderm has been observed to form as a third layer between endoderm and ectoderm, but, in the majority of cases, ectoderm attenuation occurs and mesoderm apparently forms by some mechanism such as in situ differentiation, which is not typical of the embryo in utero.
a Based on the observations of Wiley et al. (submitted for publication). * Parietal endoderm does form in cultures of isolated ICMs under some conditions 1976; Solter, personal communication).
The difference in the type of endoderm present may account for the difference in cell adhesiveness between ICM and teratocarcinoma embryoid bodies cultured in vitro bee Table 1). Also, the almost-complete absence of visceral endoderm in the cultures of cells which form only simple embryoid bodies (SCC-S2 cells) might account for their inability to progress beyond the two-layer stage when kept in suspension. Experiments are now in progress to attempt to alter the type of endoderm formed by the SCC-S2 cells and to determine if this will affect the ability of the core cells to differentiate further when kept in suspension. In spite of the differences between the teratocarcinoma and embryo-derived material, the close similarity between the two makes it evident that such tumorderived cell lines can be useful for study-
(Strickland
et al.,
ing several aspects of cell determination and differentiation in the early embryo. First, since we did not observe any endodermal cells in the interior of the clumps prior to the appearance of endoderm on the outer surface, these results are consistent with the premise that it is the location of the cells on the outer surface of the teratocarcinoma clumps that is the determining factor in their differentiation to endoderm (Martin and Evans, 1975a,b). A similar role for positional information in the determination of endoderm on the surface of the embryonic ICM has been suggested by the results of Rossant (1975) and of Solter and Knowles (1975). We have demonstrated the presence of undifferentiated cells on the outer surface of teratocarcinoma clumps and observed what appear to be the earliest changes in the differentiation to endoderm, the formation
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of junctional complexes between the cells. formation of a third layer from a primitive Although we had expected that, prior to streak. In both studies, the very few cases detachment from the tissue culture sur- in which mesoderm clearly formed as a face, the cells would be uniformly undifferthird layer did not account for the number structures that contained entiated, we found that this was not the of balloon-like It is case: At the time of detachment, the mesoderm-like cells and capillaries. clumps often had a few individual endo- not clear from either study what that dermal cells or a crescent of endoderm on mechanism of mesoderm formation is in the surface, but never a complete outer these cases. However, one possibility layer. Such results are, however, not sur- which is suggested by the observations is prising; if positional information does play that mesoderm arises by in situ differena role in endoderm formation, we might tiation of attenuated ectoderm. In conclusion, the observations reported expect to find some endoderm formed over the free surface of the clumps while they here emphasize the usefulness of these were still attached, but none formed where clonal teratocarcinoma stem cells, which the flattened clumps contacted the culture can be maintained in the undifferentiated substratum. state or used to produce at will very large Second, analysis of the next stage in numbers of cystic embryoid bodies that develop in vitro in the manner described teratocarcinoma differentiation, cavitation of the core, may also give us some here. Such cells can thus provide a model system for the study of the role of posiinsight into the process of cell determinain the processes of ention. During the development of the cav- tional information doderm formation and cavitation, and also ity, we observed a great deal of cellular for studies of the basic underlying mechadebris, presumably a result of cell death. It is unlikely that this was due to nonspe- nism of mesoderm formation, as well as other aspects of early embryonic developcific effects, since, under the same condiment. tions, the core cells of simple embryoid bodies remain healthy for 10 days or more. We would like to thank Dr. S. Rapasardi and Furthermore, cavitation in embryoid bod- Ms. Kitty Wu for technical advice and assistance, and Drs. R. Pedersen, P. Calarco, and G. S. Martin ies always appeared to occur at a distance of two or three cell diameters from the for helpful discussion and criticism during the preparation of this manuscript. We are particularly surface of the embryoid body. This localgrateful to Mr. D. Akers for his expert help in ized cell death is consistent with the sug- preparing the photographic material. gestion of Bonnevie (1950) that the proamThis work was supported in part by Special Grant niotic cavity of the mouse embryo may be No. 854 (to GRM) from the California Division, American Cancer Society, and by the U.S. Energy formed by “programmed cell death.” That is, the cells which are at the center of the Research and Development Administration. REFERENCES ICM are in some way programmed to die. Positional information may again play a BONNEVIE, K. (1950). New facts on mesoderm formation and proamnion derivatives in the normal role in this developmental event. Since mouse embryo. J. Morphol. 86, 495-545. the embryoid bodies appear to undergo a DAMJANOV, I., and SOLTER, D. (1974). Experimental process of cavitation similar to proamteratoma. Cur. Top. Pathol. 59, 69-130. niotic cavity formation in the embryo, they DAMJANOV, I., SOLTER, D., BELICZA, M., and SKREB. N. (1971). Teratomas obtained through extrautermight provide a suitable model system in ine growth of seven-day mouse embryos. J. Nat. which to examine the role of cell death Cancer Inst. 46, 471-480. during cavitation in the normal embryo. DIWAN, S. B., and STEVENS, L. C. (1976). DevelopFinally, from our observations and those ment of teratomas from the ectoderm of mouse of Wiley et al., it appears that mesoderm egg cylinders. J. Nat. Cancer Inst. 57, 93’7-942. EVANS, M. J. (1972). The isolation and properties of can arise by some mechanism other than
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a clonal tissue culture strain of pluripotent mouse teratoma cells. J. Embryol. Exp. Morphol. 28, 163-173. EVANS, M. J., and MARTIN, G. R. (1975). The differentiation of clonal teratocarcinoma cell cultures in vitro. In “Roche Symposium on Teratomas and Differentiation” (M. Sherman and D. Solter, eds.), pp. 237-250. GARDNER, R. L., and PAPAIOANNOU, V. E. (1975). Differentiation of the trophectoderm and inner cell mass. In ‘%arly Development of Mammals” (M. Balls and A. E. Wild, eds.), pp. 107-132. Cambridge University Press, Cambridge. GRAHAM, C. (1977). Teratocarcinoma cells and normal mouse embryogenesis. In “Concepts in Mammalian Embryogenesis” (M. Sherman, ed.). MIT Press, Cambridge, Mass. (in press). Hsu, Y. C., and BASKAR, J. (1974). Differentiation in vitro of normal mouse embryos and mouse embryonal carcinoma. J. Nat. Cancer Inst. 53, 177-185. ILLMENSEE, K., and MINTZ, B. (1976). Totipotency and normal differentiation of single teratocarcinoma cells cloned by injection into blastocysts. Proc. Nat. Acad. Sci. USA 73, 549-553. MARTIN, G. R. (1975). Teratocarcinomas as a model system for the study of embryogenesis and neoplasia: Review. Cell 5, 229-243. MARTIN, G. R., and EVANS, M. J. (1974). The morphology and growth of a pluripotent teratocarcinoma cell line and its derivatives in tissue culture. Cell 2, 163-172. MARTIN, G. R., and EVANS, M. J. (1975a). The differentiation of clonal lines of teratocarcinoma cells: formation of embryoid bodies in vitro. Proc. Nat. Acad. Sci. USA 72, 1441-1445. MARTIN, G. R., and EVANS, M. J. (1975b). 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” (M. Sherman and D. Solter, eds.), pp. 169-187. Academic Press, New York. MARTIN, G. R., and EVANS, M. J. (1975c). Multiple differentiation of clonal teratocarcinoma stem cells following embryoid body formation in u&o. Cell 6, 467-474. MARTIN, G. R., EPSTEIN, C. J., TRAVIS, B., TUCKER, G., YATZIV, S., MARTIN, D. W., JR., CLIP, S., and COHEN, S. X-chromosome inactivation during differentiation of female teratocarcinoma cells in vitro (submitted for publication).
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B., and ILLMENSEE, K. (1975). Normal genetically mosaic mice produced from malignant teratocarcinoma cells. Proc. Nat. Acad. Sci. USA 72, 3585-3589. PIERCE, G. B., DIXON, F. J., and VERNEY, E. L. (1959). Testicular teratomas. I. Demonstration of teratogenesis by metamorphosis of multipotential cells. Cancer 12, 573-583. PIERCE, G. B., and VERNEY, E. L. (1961). An in vitro and in uivo study of differentiation in teratocarcinomas. Cancer 14, 1017-1029. PIERCE, G. B., and BEALS, T. F. (1964). The ultrastructure of primordial germinal cells and fetal testes and of embryonal carcinoma cells in mice. Cancer Res. 24, 1553-1567. PIERCE, G. B., STEVENS, L. C., and NAKANE, P. K. (1967). Ultrastructural analysis ofthe early development of teratocarcinomas. J. Nat. Cancer Inst. 39, 755-773. ROSSANT, J. (1975). Investigation of the determinative state of the mouse inner cell mass. II. The fate of isolated inner cell masses transferred to the oviduct. J. Embryol. Exp. Morphol. 33, 9911001. SNELL, G. D., and STEVENS, L. C. (1966). In “Biology of the Laboratory Mouse,” 2nd ed, pp. 205-245. McGraw-Hill, New York. SOLTER, D., and KNOWLES, B. (1975). Immunosurgery ofthe mouse blastocyst. Proc. Nat. Acad. Sci. USA 72, 5099-5102. SOLTER, D., BICZYSKO, W., PIENKOWSKI, M., and KOPROWSKI, H. (1974). Ultrastructure of mouse egg cylinders developed in vitro. Anat. Rec. 180, 263-280. STEVENS, L. C. (1959). Embryology of testicular teratomas in strain 129 mice. J. Nat. Cancer Inst. 23, 1249-1295. STEVENS, L. C. (1970). The development of transplantable teratocarcinomas from intratesticular grafts of pre- and postimplantation mouse embryos. Develop. Biol. 21, 364-382. STRICKLAND, S., REICH, E., and SHERMAN, M. (1976). Plasminogen activator in early embryogenesis: Enzyme production by trophoblast and parietal endoderm. Cell 9, 231-240. WILEY, L. M., and PEDERSEN, R. A. (1977). Morphology of mouse egg cylinder development in vitro: A light and electron microscopic study. J. Exp. Zool. 200, 389-402. WILEY, L. M., SPINDLE, A., and PEDERSEN, R. Development of isolated mouse inner cell masses in vitro (submitted for publication). MINTZ,
BIOLOGY
61, 230-244 (197’i’l
The Development
of Cystic Embryoid Bodies in Vitro from Clonal Teratocarcinoma Stem Cells R. MARTIN’
GAIL Department
of Anatomy
and Cancer Research Institute, University 94143
of Radiobiology,
University
San Franciscd,
California
M. WILEY~
LYNN Laboratory
of California,
of California,
San Francisco,
California
94143
AND IVAN Department
of Pathology, Received April
University
DAMJANOV~ of Connecticut,
Farmington,
Connecticut
06032
20, 1977; accepted in revised form July 27, 1977
Certain clonal teratocarcinoma stem cell lines can be maintained in the undifferentiated state or can be stimulated to differentiate by a simple alteration in the culture conditions. In these cultures, differentiation occurs in a clearly definable, relatively synchronous sequence of events, as the entire cell population participates in the process of “cystic embryoid body” formation. We have studied the morphological changes that occur during this process. The pattern of development in the embryoid bodies is remarkably similar to that in normal mouse embryogenesis up to the time of formation of the third germ layer, the mesoderm. Whereas, in some embryoid bodies, mesoderm appears to form in a relatively normal manner, in moat it apparently arises by a process which has not previously been described. These results are discussed in comparison with similar studies on the development of isolated mouse inner cell masses in vitro. INTRODUCTION
The close similarity between mouse embryonal carcinoma cells, which are the stem cells of the tumors known as teratocarcinomas, and the cells of the early embryo has been dramatically demonstrated by the results of Mintz and Illmensee (1975; Illmensee and Mintz, 1976). In their experiments, embryonal carcinoma cells injected into mouse blastocysts were ’ Send reprint requests to Gail Martin, Department of Anatomy, University of California, San Francisco, California 94143. * Present address: Department of Anatomy, University of Virginia, Charlottesville, Virginia 22901. 3 Present address: Hanemann Medical College, 230 N. Broad Street, Philadelphia, Pennsylvania 19103.
found to participate with the host blastocyst cells in the formation of normal chimerit animals, which were born after the operated embryos were transferred to foster mothers. Not only were single embryonal carcinoma cells found to form a variety of somatic tissues, but also the tumor stem cells formed functional sperm in the chimeric animals. Thus, like the cells of the early embryo, embryonal carcinoma cells are totipotent. That is, they are capable of multiplying in the undifferentiated state and of giving rise to all of the differentiated cell types in an animal. The “embryonic” nature of embryonal carcinoma cells has also been demonstrated in other kinds of experiments. For example, when a solid teratocarcinoma is 230
Copyright 0 1977by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN
0012-1606
MARTIN,
WILEY,
AND DAMJANOV
injected into the peritoneal cavity of an adult mouse, the pluripotent stem cells can form embryonic structures known as “embryoid bodies” (Stevens, 1959; Pierce et al., 1959). These bear a superficial resemblance to mouse blastocysts of approximately 3.5days gestation, and are sometimes mistaken for them. However, in the blastocyst, the outer cell layer is trophectoderm surrounding an inner cell mass (ICM), while the embryoid bodies have an outer layer of endoderm surrounding an embryonal carcinoma core and do not contain any trophoblast derivatives. Such embryoid bodies are therefore more directly comparable to the inner cell mass derivatives of a normal embryo of approximately 5-days gestation, when the ICM has developed an outer layer of endoderm. These two-layered teratocarcinoma derivatives, known as “simple” embryoid bodies, will sometimes develop in the peritoneal cavity into more complex “cystic” embryoid bodies, which have numerous similarities to older embryos. It has also been shown that simple embryoid bodies removed from the peritoneal cavity can differentiate into cystic embryoid bodies in vitro and that this process parallels normal embryonic development (Pierce and Verney, 1961; Hsu and Baskar, 1974). The advantages of using the tumor stem cells as a substitute for the cells of the normal early embryo in many kinds of experimental studies have long been recognized. However, the usefulness of transplantable tumor lines is limited by the heterogeneity of the cell population and by the exposure of the cells to indeterminate influences in the host environment. Both of these limitations have been overcome since undifferentiated teratocarcinoma stem cells have been cloned and cultured in vitro (reviewed by Damjanov and Solter, 1974; Martin, 1975). The potential usefulness of such pluripotent cell lines, however, is to a certain degree dependent on their ability to differentiate in vitro in a manner that parallels normal
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embryogenesis. Most of the embryonal carcinoma cell lines that are available have not been reported to mimic normal embryonic morphogenesis to any significant extent in the course of their differentiation, although some of these cell lines can form derivatives of all three germ layers after several weeks of culture under the appropriate conditions (see review by Graham, 1977). In contrast, the clonal embryonal carcinoma cell cultures isolated by Martin and Evans (1975a,b) differentiate in a specific sequence of events, as the entire population participates in the relatively synchronous formation of embryoid bodies. These embryonal carcinoma cells are maintained in the undifferentiated state by frequent subculture in the presence of fibroblastic “feeder” cells: When passaged in the absence of feeder cells, the embryonal carcinoma cells form rounded aggregates, and differentiation begins with the formation of a layer of endodermal cells over the whole outer surface of these aggregates. Some lines of these clonal embryonal carcinoma cells stop their differentiation at this stage and remain as two-layered, simple embryoid bodies when kept in suspension; However, Martin and Evans (1975a,b) have noted that, under the same conditions, other clonal teratocarcinoma stem cell lines will continue to differentiate to complex, cystic embryoid bodies. Since such clonal cell cultures are being used as an in vitro model system for studying various aspects of early embryonic development, such as X-chromosome inactivation (Martin et al ., submitted for publication), the primary objective of the present study was to analyze in some detail the process of cystic embryoid body formation by these teratocarcinoma stem cell lines. In so doing, we hoped to determine the extent to which these cells, like the transplantable tumor lines passaged in ho, differentiate in a pattern that resembles the early postimplantation development of the mouse embryo. In making the
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DEVELOPMENTAL BIOLOGY
comparison between embryoid body formation and normal embryogenesis, we have paid particular attention to the development in vitro of isolated mouse ICMs, as described by Wiley et al. (submitted for publication); such a comparison is highly relevant, since both the embryoid bodies and the isolated ICMs begin their development with the formation of an outer layer of endoderm, and, unlike the situation in the intact embryo, differentiation continues in the absence of trophoblastic tissue. MATERIALS
AND
METHODS
All clonal embryonal carcinoma cell lines were derived from isolated single cells as previously described (Martin and Evans, 1975a, b). Unless otherwise noted, the culture medium used in these experiments was Dulbecco’s modified Eagle’s medium (containing 4.5 g/liter of glucose) supplemented with 10% calf serum (Gibco, selected batches). Cultures were incubated in 5% CO* (in air) at 37°C. The clonal cell line PSAl forms embryoid bodies that become cystic when kept in suspension. It was isolated from secondary cultures of transplantable tumor OTT 5568, which was initiated by the transfer of a 3-day mouse embryo to an ectopic site (Stevens, 19701. The cell line SCC-S2 forms embryoid bodies that remain simple when kept in suspension. It is a clonal derivative of the main stock of the SIKR teratocarcinoma cell line, also derived from tumor OTT 5568, which has been previously described (Evans, 1972; Martin and Evans, 1974, 1975b). Stock cultures of the two pluripotent embryonal carcinoma cell lines were maintained in the undifferentiated state by subculturing the cells every 3 days and seeding them on a confluent feeder layer of Mitomycin C-treated ST0 mouse cells (Martin and Evans, 1975a). To induce embryoid body formation, the cells were seeded at lo7 cells per g-cm tissue culture dish. On the third day after
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this passage, in the absence of feeder cells, clumps of cells had formed. These were then detached from the dish by gently pipetting fresh medium over them. The detached clumps were allowed to settle for several minutes, washed in fresh medium, and then seeded in a bacteriological petri dish. The medium was changed daily during the subsequent 11-day culture period of the cells in suspension. At the time of detachment, and at 12- or 24-hr intervals thereafter, an aliquot of several hundred cell clumps was fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, and subsequently postfixed with 2% 0~0, in the same buffer. The cells were dehydrated in a graded series of ethanol in water, during which they were stained en bloc with uranyl acetate. After complete dehydration by passage through propylene oxide the clumps were embedded in Epon resin and sectioned for either light or transmission electron microscopy, as previously described (Wiley and Pedersen, 1977). RESULTS
Cystic Embryoid Body Formation Observed in the Phase-Contrast Microscope
as
In order to initiate cystic embryoid body formation, PSAl cells were plated on tissue culture dishes in the absence of feeder cells, at a time designated as Day 0 (D,). After 3 days of culture (D3), the cell clumps which had formed were detached and plated (at a time designated as D, + J in bacteriological petri dishes to which they do not adhere. The changes that occurred in the clumps over an 11-day culture period in suspension (D3 + 1, D, + 2, etc.) were followed by phase-contrast microscopy. At the time of detachment, the rounded PSAl embryonal carcinoma cell clumps appeared to be homogeneous with respect to cell type and had a smooth, refractile surface (Fig. 1A). A small fraction of the
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FIG. 1. Cystic embryoid body formation, as observed in the phase-contrast microscope. (A) Clumps of embryonal carcinoma cells at the time of detachment from the tissue culture substratum (D, + ,J. Approx. 200 x . (B) After 2 days in suspension CD, + 2) the embryonal carcinoma cell clumps have become simple embryoid bodies. Pointers indicate the outer layer of endoderm. Approx. 200 X. (0 By D, + B a cavity is apparent in most of the embryoid bodies. Approx. 200 X. (D) A large proportion of the embryoid bodies develops balloon-like cysts by Day 10 in suspension. Approx. 90 x .
clumps, however, did have some obvious endodermal cells on their outer surface. Within 24 hr of detachment (II3 + J, all of the clumps had an outer layer of endodermal cells surrounding an inner cell core and had thus become simple embryoid bodies. There was an apparent increase in clump size, probably due both to clump aggregation and to cell multiplication. By 48 hr after detachment (II3 + 2), the endodermal cell layer of these embryoid bodies had become more clearly delineated (Fig. 1B); no other changes were apparent.
At 4 days after detachment (OS + J most of the embryoid bodies were still composed of a solid core surrounded by a layer of endoderm, although in a small fraction of the population a cavity was apparent. By 5 to 6 days after detachment, most of the embryoid bodies showed internal changes: The cells had become arranged around what appeared to be an eccentrically placed cavity (Fig. 1C). After 6 days in suspension, some of the embryoid bodies began to expand and to show a balloonlike swelling on one side. Over the next
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few days, a large proportion of the embryoid bodies developed similar balloonlike cysts; these structures continued to enlarge and often reached several millimeters in diameter (Fig. 1D). Although the more solid portion of the embryoid bodies usually remained attached to the balloon-like cyst, it often appeared to atrophy, and in some cases to disappear altogether. Structural Analysis of the Process of Cystic Embryoid Body Formation In order to compare the internal changes that occurred during the process of cystic embryoid body formation with those that occur during development of the early embryo, aliquots of the embryoid bodies were fixed at various times after detachment, embedded, and sectioned. Approximately 50 embryoid bodies were examined at each time point. Although the morphological features described here characterized the embryoid bodies as a population, it should be noted that the progression of the population through each successive stage in development was not perfectly synchronous. (A) Clump structure at the time of detachment. Light-microscopic examination of the cell clumps fixed at the time of detachment indicated that they were not as morphologically homogeneous as they had appeared in the phase-contrast microscope. Although some clumps did consist of only embryonal carcinoma cells, in most there were one or more endodermal cells on the surface of the clump. In a few cases, a crescent of endodermal cells was present (Fig. 2). However, in none of the clumps fixed at the time of detachment did there appear to be a complete layer of endodermal cells encircling the embryonal carcinoma cells. Similar results were obtained when clumps were detached at an earlier time (i.e., D, + ,,, 2 instead of 3 days after plating). The structure of the endodermal cells that were present at the
FIG. 2. Clump of embryonal carcinoma cells shortly after detachment from the substratum. Pointers indicate a crescent of endodermal cells. Approx. 400 x.
time of detachment of the clumps is described below [Section (B)]. Our observations on the fine structure of the embryonal carcinoma cells correspond with previously published descriptions of such cells (Pierce and Beals, 1964; Pierce et al., 1967; Damjanov et al., 1971). They had large, irregularly shaped nuclei that contained two or three large nucleoli (Fig. 3A). Euchromatin predominated over heterochromatin, although numerous dense bodies were scattered throughout the nucleoplasm. The cytoplasm contained many free ribosomes, a few small spherical mitochondria, occasional profiles of nondistended rough endoplasmic reticulum, moderately developed Golgi, and a few membrane-bound dense bodies. We found that intracisternal A particles were not readily apparent. At the time of detachment, the embryonal carcinoma cells were closely packed and there were occasional intercellular clefts (Fig. 3A). Although a few cell junctions were present, they looked rather primitive and resembled zonulae adherents. No intercellular material was found between the cells. Those embryonal carcinoma cells on the outer surface of the clumps were mostly cuboidal, with ran-
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FIG. 3. Fine structural details of the cells present at the early stages of embryoid body formation in uitro. 4105 x (A) Undifferentiated embryonal carcinoma cells. The nucleus (N) contains prominent nuclei (n) and numerous dense bodies. (B) Cuboidal cell at the surface of a clump at D3 + 1. This cell contains profiles of rough endoplasmic reticulum (rer) distended with a flocculent material, and is joined to its neighbor by apical cell junctions (arrow). (C) Parietal endoderm cell. The cytoplasm contains distended profiles of rer, as well as some extracellular material that resembles Reichert’s membrane km) along its basal aspect. (D) Visceral endoderm cell. Many apical microvilli and electron-lucent vacuoles (VI distinguish this cell type from parietal endoderm cells and from embryonal core cells.
domly distributed organelles and no apical microvilli or vacuoles. (B) Morphological features of endodermal cells. The endodermal cells of embryoid bodies were of two distinct types, corresponding to the two types of primary endoderm found in the mouse embryo. One resembled parietal (distal) endoderm, as distinguished by its distended profiles of rough endoplasmic reticulum (RER; Figs. 3B and C). Within these dilated RER, and also as a thick layer underlying these parietal endodermal cells, was a substance thought to be analogous to the mucoprotein Reichert’s membrane of the mouse embryo. The second cell type re-
sembled visceral (proximal) endoderm, as identified by its narrow profiles of RER, numerous apical electron-lucent vacuoles, and numerous microvilli (Fig. 3D). Reichert’s membrane-like material was also occasionally observed underlying these visceral endodermal cells. Both types of endoderm were present in roughly equal proportions, although one or the other type often predominated on an individual embryoid body. The distinction between the two cell types, however, was not always clear-cut, and occasional, intermediate forms were observed. During the 24-36 hr that followed detachment of the clumps, the endodermal
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cells formed a continuous layer around the clumps. Endoderm formation appeared to begin with the flattening of outer cells, which was accompanied by the formation of apical junctions and an increase in dilated RER, so that the cells resembled parietal endoderm (Figs. 3B and 0. Shortly after the appearance of these features, cells that had the characteristics of visceral endoderm were also apparent in the outer cell layer. From our observations, it is not possible to say whether these arose from cells initially resembling parietal endoderm or whether they arose directly from the embryonal carcinoma
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cells. Interestingly, intracisternal A particles were common in these visceral endoderm cells, but were not apparent in parietal endoderm. (C) Cavitation and formation of a columnar epithelium. About 4 to 5 days after detachment, internal changes in the cores of the embryoid bodies became apparent. The first change was the appearance of one or more small cavities full of cellular debris (Fig. 4A). These usually formed two to three cell diameters from the outer surface; since the embryoid bodies were often more than 10 cells in diameter, the cavity therefore formed eccentrically, thus
FIG. 4. Cavitation and formation of a columnar epithelium in embryoid bodies. (A) First appearance of a cavity at approximately D, + ,. Two or three cell diameters from the outer endodermal cell layer (en), a small cavity (cav) filled with cellular debris becomes apparent. Approx. 400 X. (B) Detail of core cells surrounding debris-filled cavity shown in Fig. 4A. These cells are joined by apical cell junctions (arrows). 4105 X. (Cl Shortly after its initial appearance, the cavity was enlarged and the cells lining it have elongated and formed a columnar epithelium. Approx. 400 x. (D) Typical population of cavitated embryoid bodies at D, + ,. Approx. 100 x.
MARTIN, WILEY, AND DAMJANOV
establishing polarity in the embryoid bodies. The embryonal carcinoma cells surrounding this cavity had formed junctional complexes bordering the cavity (Fig. 4B), while embryonal carcinoma cells elsewhere in the cores of the embryoid bodies were no different from the undifferentiated cells at the time of detachment and showing only unspecialized junctions between them. At the time that the cavity formed, or shortly thereafter, the embryonal carcinoma cells that surrounded the cavity elongated to create a simple epithelium of columnar cells (Fig. 40. This process is analogous to the formation of the proamniotic cavity and embryonic ectoderm in the normal mouse embryo. These changes were relatively synchronous in the population as a whole, and all of the embryoid bodies developed cavities enclosed by a layer of columnar cells (Fig. 4D). The apparent heterogeneity of the population in Fig. 4D is probably a consequence of variation in the plane of section through these asymmetric embryoid bodies. (0) Mesoderm formation in cystic embqoid bodies. In the normal development of the embryo, mesoderm differentiation follows the formation of the proamniotic cavity and columnar ectodermal epithelium: A primitive streak, or thickening, on one side of the embryonic ectoderm is established and mesodermal cells then delaminate into the space between the ectoderm and the endoderm, thus forming a third layer of cells (see Snell and Stevens, 1966). Evidently, mesoderm could form in an analogous way in the embryoid bodies, since we found at least two clear examples of this (out of approximately 200 embryoid bodies examined at this stage), as shown in Fig. 5. In these embryoid bodies, a third layer of loosely arranged cells was found between the endoderm and the ectoderm. Observations of these cells in the transmission electron microscope indicated that they did not form junctional complexes with one another (Fig. 5B), in
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contrast to the cells in both the endodermal and ectodermal cell layers, which usually did. The cells in this third layer resembled mesoderm, in that they were oriented lengthwise in the cleft between the endoderm and ectoderm, lying outside of any basal lamina that was present under the ectoderm or endoderm. Also, like mesodermal cells in intact cultured embryos (Wiley and Pedersen, 19771, they contained fusiform nuclei and had many free ribosomes, but few that were membrane bound. Although mesoderm formation apparently occurred in most of the embryoid bodies, it did not necessarily arise by a process analogous to normal development. Except for the two examples described above, we found little evidence of mesoderm delamination. Between D, + 6 and D 3 + 109 many embryoid bodies underwent expansion of the cavity and attenuation of their columnar epithelial cells (Fig. 6A). The cells lining the cavity thus came to resemble the mesodermal cells which were subsequently observed in the cultures (see below). Many of the balloon-like structures which resulted from this cavity expansion and cell attenuation retained a focus of morphologically undifferentiated embryonal carcinoma cells at one pole (Fig. 6B). (At first glance, this particular example looks misleadingly like a mouse blastocyst, but since the wall of the cyst is two cell layers thick, the outer one of which is visceral endoderm, this structure is in no way analogous to a blastocyst.) In some embryoid bodies, the cells underlying the endoderm appeared to indent into the cavity (Fig. 60. By lo-11 days in suspension, the population of embryoid bodies had become considerably more pleiomorphic and was found to contain complex double cysts of the type shown in Fig. 7, in addition to the single cysts described above. Our observations did not provide any clear indication of how the double cysts might have arisen from the less-complex structures.
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FIG. 5. Mesoderm formation in embryoid bodies. (A) Between the outer endoderm (en) and the inner ectodermal layer fee) is a third layer of fusiform cells which resemble mesoderm (mes). Approx. 400 x (B) Ultrastructure of the embryoid body shown in Fig. 6A. A mesodermal cell (mea) occupies the cleft between the endoderm (en) and ectoderm (ec). 5941 x. (C) Another example of a primitive streak-like area. Approx. 400 x.
Such double cysts were enclosed by one continuous layer of endoderm and contained two inner vesicles, one of which was expanded and attenuated, while the other resembled the unexpanded columnar ectodermal vesicle present earlier in the culture period (Figs. 7A and B). In some cases, however, this unexpanded vesicle appeared to have atrophied (Figs. 7C and D) or even disappeared altogether. Structures which resembled blood-filled capillaries were often observed in the attenuated cell layer lining the balloon-like cyst walls (Fig. 7E), thus suggesting that
the mesoderm lining the cyst walls arose from the attenuated cells. These balloonlike cysts were remarkably similar to the yolk sac which develops in the embryo between 7 and 8 days of gestation. In none of the embryoid bodies that we examined did we observe any cells which resembled extraembryonic ectoderm or its derivatives. This is not surprising, since the extraembryonic ectoderm is thought to be a trophoblast derivative (Gardner and Papaioannou, 1975) and there is no evidence that embryoid bodies ever contain any trophoblastic cells. We also did
MARTIN,
WILEY,
AND
FIG. 6. Ectoderm attenuation in cyst and apparently attenuated inner attenuated ectoderm and a focus of indentation of ectoderm (ie). Approx.
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expanding cystic embryoid bodies. (A) Embryoid body with a single layer of ectoderm at D, + 1,,. Approx. 400 x. (B) Embryoid body with undifferentiated cells (u). Approx. 150 X. (C) Embryoid body with 150 x
not observe any clear examples of “paired cysts” of the type frequently formed by isolated ICMs cultured in vitro (Wiley et Neither al., submitted for publication). did we observe the “budding” leading to polycyst formation which occurred in the cultures of embryoid bodies removed from the peritoneal cavity (Hsu and Baskar, 1974). Studies of Teratocarcinoma Stem Cells that Form Only Simple Embryoid Bodies As noted above, certain clonal embryonal carcinoma cell lines form embryoid bodies which remain simple when kept in suspension (Martin and Evans, 1975a, b). These cells, like those that form cystic embryoid bodies, can, however, differentiate to a variety of cell types such as
cartilage and keratinizing epithelium if the embryoid bodies are allowed to attach to a substratum (Martin and Evans, 1975c; Evans and Martin, 1975). We have compared the behavior in suspension culture of one such cell line, SCC-S2, with that of the PSAl cells that form cystic embryoid bodies, in the hope of gaining some insight into the cause of the differences in their differentiative patterns. In the undifferentiated state, there were no apparent morphological differences between the two cell types. Under conditions that allow development into embryoid bodies, however, we observed one striking difference between them. In contrast to the cystic embryoid bodies, which had a mixture of both visceral and parietal endoderm, virtually all of the endoderm on
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FIG. 7. Features of balloon-like cysts present in the embryoid body population by D, + ,O. (A) Detail of ectodermal vesicle (ec) present in a double cyst. Note the single layer of endodermal cells (en) surrounding both cavities and the single attenuated cell layer lining the larger cavity. Approx. 400 x. (B) Low magnification of the double cyst shown in Fig. 7A. Approx. 100 X. (C) Another double cyst, with a somewhat atrophied ectodermal vesicle (ec). Approx. 100 X. (D) Detail of the ectodermal vesicle of the double cyst shown in Fig. 7C. Approx. 300 x (El Typical capillary-like structure found in the attenuated cell layer of the balloon walls. Approx. 300 x.
the simple embryoid bodies formed by the SCC-S2 cells was of the parietal type. This probably explains the observation that the cores of these embryoid bodies rapidly became encased in a very thick layer of Reichert’s membrane-like material and thus after several days in suspension the endodermal cells often detach from the outer surface. We examined approximately
200 simple embryoid bodies at various times in the course of the experiment, and on only one occasion did we observe a small number of outer cells of SCC-S2 embryoid bodies that was clearly of the visceral type. This difference in the type of endoderm formed probably accounts for the earlier observation (Martin and Evans, 1975c) that, when simple and cystic em-
MARTIN, WILEY, AND DAMJANOV
bryoid bodies attached to a substratum, the endodermal cells which migrated away from the cores of these two types were morphologically different from one another. DISCUSSION
The observations described here demonstrate the similarity in the fundamental pattern of development of cystic embryoid bodies formed in vitro from clonal teratocarcinoma stem cells and of the ICM of the mouse embryo; in both cases, the first differentiative event is the formation of an outer endodermal cell layer. This is followed by cavitation and subsequent formation of a columnar epithelium by the inner cells. At the stage of mesoderm formation, however, there is an apparent deviation from the normal pattern of development in the embryoid bodies. Although mesoderm can form as a third layer between the endoderm and ectoderm, in most cases it appears to arise by some other process. That this abnormal method of mesoderm formation is not an anomaly intrinsic to teratocarcinoma cells, however, has been clearly demonstrated by the work of Wiley et al. (submitted for publication), which showed that, in the large majority of isolated inner cell masses cultured in vitro, mesoderm also apparently develops by some method other than formation of a third layer of cells. This latter observation underscores the need for comparing the development of embryoid bodies with that of the isolated ICM in vitro, rather than with the behavior of the intact embryo. In both the embryoid bodies and the isolated ICM cultured in vitro, development proceeds independently of trophoblast-derived tissues, although one cannot rule out the possibility that contact with the trophoblast prior to isolation of the ICM might have some determinative effects on the cells of the ICM. Such a comparison is shown in Table
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1, which is based on our observations and those of Wiley et al. While the similarities between the development of cystic embryoid bodies and isolated ICMs cultured in vitro are striking, there are also differences. One of these concerns the type of endodermal cells present. In the normal embryonic egg cylinder in utero and also in the experiments of Wiley et al ., the endoderm covering the ICM and its derivatives is the visceral type only. Thus, a thick layer of Reichert’s membrane, a parietal endoderm product, is not observed between the embryonic ectodermal epithelium and the endoderm. In contrast, in our cultures, the cystic embryoid bodies always contained a high proportion of parietal endoderm, as evidenced by the thick layer of Reichert’s membrane-like material between the inner columnar epithelium and the outer endodermal cell layer, shown in Fig. 4C. Experiments designed to determine if differences in the culture medium employed in this and the study by Wiley et al. were responsible for the type of endoderm formed indicated that this was not the case. However, cells of the parietal type have been reported to form part of the endodermal layer covering ICM derivatives in both intact embryonic egg cylinders cultured in vitro (Solter et al. 1974) and isolated inner cell masses cultured in vitro (Strickland et al., 1976; Solter, personal communication). At present, there is no explanation of why in some cases embryonic material cultured in vitro apparently produces a mixture of parietal and visceral endoderm and in other cases it does not. However, the fact that cells with features of both types of endoderm can be found in our cultures suggests that the two endodermal cell types may be closely related. This idea is supported by the observation of Diwan and Stevens (1976) that isolated visceral endoderm grafted to the testes of adult mice can produce large amounts of Reichert’s membrane.
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COMPARISON OF TERATOCARCINOMA CYSTIC EMBRYOID BODY FORMATION WITH MOUSE INNER CELL MASS DEVELOPMENT in Vitro Stage of development Endoderm
Cavitation columnar
in vitro
formation
and formation epithelium
of a
Features of teratocarcinoma tic embryoid bodies
cys-
formation
of isolated
ICMs”
Endodermal cell layer is a mixture of both visceral and parietal types. Readily attach to a tissue culture substratum at all stages of development.
Endodermal cell layer is almost exclusively of the visceral type. b Little or no attachment to a tissue culture substratum after endoderm formation.
Cavity
Cavity forms centrally and all of the inner cells form a columnar epithelium surrounding the cavity.
formation
may be eccentric.
Columnar epithelium forms around the cavity, but, in cases where the cavity is eccentric, the columnar epithelium does not completely surround the cavity and some cells apparently remain undifferentiated. Mesoderm
Features
Mesoderm has been observed to form as a third layer between endoderm and ectoderm, but, in the majority of cases, ectoderm attenuation occurs and mesoderm apparently forms by some mechanism such as in situ differentiation, which is not typical of the embryo in utero.
a Based on the observations of Wiley et al. (submitted for publication). * Parietal endoderm does form in cultures of isolated ICMs under some conditions 1976; Solter, personal communication).
The difference in the type of endoderm present may account for the difference in cell adhesiveness between ICM and teratocarcinoma embryoid bodies cultured in vitro bee Table 1). Also, the almost-complete absence of visceral endoderm in the cultures of cells which form only simple embryoid bodies (SCC-S2 cells) might account for their inability to progress beyond the two-layer stage when kept in suspension. Experiments are now in progress to attempt to alter the type of endoderm formed by the SCC-S2 cells and to determine if this will affect the ability of the core cells to differentiate further when kept in suspension. In spite of the differences between the teratocarcinoma and embryo-derived material, the close similarity between the two makes it evident that such tumorderived cell lines can be useful for study-
(Strickland
et al.,
ing several aspects of cell determination and differentiation in the early embryo. First, since we did not observe any endodermal cells in the interior of the clumps prior to the appearance of endoderm on the outer surface, these results are consistent with the premise that it is the location of the cells on the outer surface of the teratocarcinoma clumps that is the determining factor in their differentiation to endoderm (Martin and Evans, 1975a,b). A similar role for positional information in the determination of endoderm on the surface of the embryonic ICM has been suggested by the results of Rossant (1975) and of Solter and Knowles (1975). We have demonstrated the presence of undifferentiated cells on the outer surface of teratocarcinoma clumps and observed what appear to be the earliest changes in the differentiation to endoderm, the formation
MARTIN,
WILEY,
AND DAMJANOV
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Differentiation
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of junctional complexes between the cells. formation of a third layer from a primitive Although we had expected that, prior to streak. In both studies, the very few cases detachment from the tissue culture sur- in which mesoderm clearly formed as a face, the cells would be uniformly undifferthird layer did not account for the number structures that contained entiated, we found that this was not the of balloon-like It is case: At the time of detachment, the mesoderm-like cells and capillaries. clumps often had a few individual endo- not clear from either study what that dermal cells or a crescent of endoderm on mechanism of mesoderm formation is in the surface, but never a complete outer these cases. However, one possibility layer. Such results are, however, not sur- which is suggested by the observations is prising; if positional information does play that mesoderm arises by in situ differena role in endoderm formation, we might tiation of attenuated ectoderm. In conclusion, the observations reported expect to find some endoderm formed over the free surface of the clumps while they here emphasize the usefulness of these were still attached, but none formed where clonal teratocarcinoma stem cells, which the flattened clumps contacted the culture can be maintained in the undifferentiated substratum. state or used to produce at will very large Second, analysis of the next stage in numbers of cystic embryoid bodies that develop in vitro in the manner described teratocarcinoma differentiation, cavitation of the core, may also give us some here. Such cells can thus provide a model system for the study of the role of posiinsight into the process of cell determinain the processes of ention. During the development of the cav- tional information doderm formation and cavitation, and also ity, we observed a great deal of cellular for studies of the basic underlying mechadebris, presumably a result of cell death. It is unlikely that this was due to nonspe- nism of mesoderm formation, as well as other aspects of early embryonic developcific effects, since, under the same condiment. tions, the core cells of simple embryoid bodies remain healthy for 10 days or more. We would like to thank Dr. S. Rapasardi and Furthermore, cavitation in embryoid bod- Ms. Kitty Wu for technical advice and assistance, and Drs. R. Pedersen, P. Calarco, and G. S. Martin ies always appeared to occur at a distance of two or three cell diameters from the for helpful discussion and criticism during the preparation of this manuscript. We are particularly surface of the embryoid body. This localgrateful to Mr. D. Akers for his expert help in ized cell death is consistent with the sug- preparing the photographic material. gestion of Bonnevie (1950) that the proamThis work was supported in part by Special Grant niotic cavity of the mouse embryo may be No. 854 (to GRM) from the California Division, American Cancer Society, and by the U.S. Energy formed by “programmed cell death.” That is, the cells which are at the center of the Research and Development Administration. REFERENCES ICM are in some way programmed to die. Positional information may again play a BONNEVIE, K. (1950). New facts on mesoderm formation and proamnion derivatives in the normal role in this developmental event. Since mouse embryo. J. Morphol. 86, 495-545. the embryoid bodies appear to undergo a DAMJANOV, I., and SOLTER, D. (1974). Experimental process of cavitation similar to proamteratoma. Cur. Top. Pathol. 59, 69-130. niotic cavity formation in the embryo, they DAMJANOV, I., SOLTER, D., BELICZA, M., and SKREB. N. (1971). Teratomas obtained through extrautermight provide a suitable model system in ine growth of seven-day mouse embryos. J. Nat. which to examine the role of cell death Cancer Inst. 46, 471-480. during cavitation in the normal embryo. DIWAN, S. B., and STEVENS, L. C. (1976). DevelopFinally, from our observations and those ment of teratomas from the ectoderm of mouse of Wiley et al., it appears that mesoderm egg cylinders. J. Nat. Cancer Inst. 57, 93’7-942. EVANS, M. J. (1972). The isolation and properties of can arise by some mechanism other than
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