Cell, Vol. 5, 229-243,

July 1975,

Copyright0

1975

by MIT

Teratocarcinomas as a Model System for the Study of Embryogenesis and Neoplasia Gail R. Martin’% Department of Anatomy and Embryology University College London Gower Street London WClE 6BT, England

Teratocarcinomas are malignant tumors which are characterized by the presence of a distinctive cell type known as embryonal carcinoma, as well as a variety of differentiated cell types, such as nerve, skin, muscle, cartilage. The embryonal carcinoma cells are the stem cell of these tumors and they show remarkable similarities to the ceils of the early embryo. The analogy between the two cell types is based on the pluripotency of the embryonal carcinoma cells, which can differentiate into a wide range of cell types, representing derivatives of all three embryonic germ layers. Teratocarcinomas can be obtained with high frequency by implantation of early embryos in extrauterine sites, which is consistent with the idea that there is a close relationship between early embryo cells and the tumor stem cells; in addition, the two cell types have at least one antigen in common, which has not been detected as yet, on other tissues except sperm. In contrast to early embryo cells, embryonal carcinoma cells are relatively easy to obtain in large numbers and to culture in vitro. Several clonal lines have been isolated, and when these cells are reinjetted into mice, they form teratocarcinomas containing a variety of cell types. Differentiation of the stem cells also can occur in vitro, and this provides an opportunity for studying cell determination, the process by which pluripotent cells become committed to a particular developmental pathway, as well as for following subsequent differentiation of the committed cells. The immunological similarity of embryonal carcinoma cells to early embryo cells may be exploited to study both the antigenic properties of embryo cells and the immunological response of the animal to them. Teratocarcinoma cells are therefore a useful alternative to embryos for the study of the processes of early mammalian development. Embryonal carcinoma cells lose their malignancy when they differentiate in vivo. This suggests that teratocarcinoma cells may also be useful for studying the relationship between neoplasia and differentiation. It is possible that the malignant behavior of the embryonal carcinoma cells is a function of their similarity to early embryo cells, and that the transition from normal pluripotent embryonic cells to malignant embryonal carcinoma cells is readily revers‘Present address: nia, San Francisco,

Department California

of Pediatrics, 94143.

University

of Califor-

Review

ible. Further study of these questions may provide proof for the hypothesis that at least some malignant changes do not involve genetic alterations. In this review I shall discuss the evidence that embryonal carcinoma cells are similar to early embryo cells and that they lose their malignancy as they differentiate. Since the relevant data obtained in studies of the tumors have already been extensively reviewed (Stevens, 1967a; Pierce, 1967; Damjanov and Solter, 1974a), emphasis here will be on recent work with teratocarcinoma cells in vitro. I hope that this discussion will help to make apparent the potential value of the cells as a model system for the study of embryogenesis and the mechanism of neoplasia. Benign and Malignant Teratomas The name of these tumors is derived from the Greek root “teraton” meaning monster, which conveys the idea of malformed development that is characteristic of them. While the composition of the tumors shows wide variation, the most typical contain derivatives of all three embryonic germ layers: ectoderm (for example, neural tissue, skin), mesoderm (for example, muscle, bone, cartilage), and endoderm (glandular structures, gut). In some cases, extra-embryonic tissues such as trophoblast and yolk sac are also present. Although some tissue organization may be found, in general the differentiated tissues appear to be chaotically arranged (Figure 1 A-E). Histological studies indicate that when they are first detectable, all teratoma tumors contain embryonal carcinoma cells (Stevens, 1959). Only those tumors in which these cells continue to multiply as undifferentiated cells are malignant. These tumors, known as teratocarcinomas, are characterized by fast growth and are retransplantable. The embryonal carcinoma cells are found haphazardly distributed throughout the tumor in the form of small groups or nests. Those tumors in which the embryonal carcinoma cells do not continue to multiply as undifferentiated cells grow more slowly, usually ceasing growth within six weeks of initiation. They are not retransplantable. Such tumors are known as benign teratomas or simply teratomas, although this term is often used to designate both the benign and malignant tumors (Stevens and Little, 1954; Stevens and Hummel, 1957). Teratocarcinomas are retransplantable, depending on the part of the tumor selected; only those portions which contain embryonal carcinoma cells will grow in the new host. Many solid tumors can be converted to growth in the ascitic form, and therefore Pierce and Dixon (1959 a,b) reasoned that by intraperitoneal injection of minced mouse teratocarcinomas it might be possible to obtain growth

Figure Typical

I. Histology tumor

of Teratocarcinomas

sections

stained

with

hematoxylin,

eosin,

and alcian

blue.

(A) A variety of differentiated tissues is apparent at low magnification. Cartilage (mesodermal derivative) is readily recognizable as the most darkly stained tissue. Magnification: X 17. (6) Early neural differentiation (ectodermal derivative). Magnification: X 100. (C) Keratinizing epithelium (skin: an ectodermal derivative). Magnification: approximately X 50. (D) Endodermal cysts. Magnification: X 130. (E) Trophoblast. Extremely large cells of extra-embryonic origin. Contrast with typical embryonal carcinoma cells (the stem cells of the tumor) shown on the left and lower right. Magnification: X 110.

Teratocarcinomas 231

Figure

2. Simple

and Embryogenesis

Embryoid

Body

Histological section (7 p) stained with hematoxylin and eosin. cells are endoderm, surrounding an inner core of embryonal noma cells. Magnification: X 520.

Figure

3. Ultrastructure

of Simple

Embryoid

Outer carci-

of only the malignant elements of these tumors. Instead of single embryonal carcinoma cells in the ascitic fluid, however, they found aggregates of cells which resembled certain stages of early mouse embryogenesis and were therefore termed embryoid bodies (Stevens, 1959, 1960; Pierce, Dixon, and Verney, 1960). Two types of embryoid body can be obtainedsimple and cystic. Simple embryoid bodies consist of an inner core of embryonal carcinoma cells surrounded by a single layer of endodermal cells (Figure 2). The outer endodermal cells can be distinguished from the inner embryonal carcinoma cells by their ultrastructural appearance (Teresky et al., 1974). They have plentiful rough endoplasmic reticulum which is swollen with a material that is also found between the outer cell layer and the embryonal carcinoma cell core (Figure 3). This mucopolysaccharide material probably corresponds to Reichert’s membrane, which is characteristically produced by endodermal cells of the

Bodies

The outer cells (endoderm, En) have characteristic features including plentiful rough endoplasmic reticulum (arrow) swollen with a product which is probably Reichert’s membrane. The inner embryonal carcinoma cells (EC) have few cytoplasmic organelles including mitochondria and numerous free ribosomes. In between the inner and outer cells is a layer of mucopolysaccharide which is probably Reichert’s membrane (RM). Magnification: X 7000. The embryoid body shown here was formed in vitro, but appears to be identical to those found in the ascitic fluid of animals bearing intraperitoneal teratocarcinomas.

Cell 232

early mouse embryo (Pierce et al., 1962; Pierce, 1966; Solter et al., 1974). The inner cells of embryonal carcinomas have few cytoplasmic organelles (a few mitochondria and sometimes Golgi complexes), and a large number of free ribosomes in the cytoplasm (Figure 3). These simple embryoid bodies resemble the embryonic portion of the 5 day mouse embryo in that the pluripotent cells are surrounded by endoderm (Figure 4~); they are often incorrectly referred to as morula-like structures. At the morula stage, however, the pluripotent cells of the embryo are surrounded not by endoderm, but by a single layer of prospective trophoblast (Figure 4a; see review by Herbert and Graham, 1974). Cystic embryoid bodies, which apparently arise from simple ones (Pierce and Dixon, 1959a), are more complex, many of them containing embryonal carcinoma cells, a variety of differentiated tissues, and a fluid-filled cyst (Figure 5). These structures bear striking similarities to the embryonic portion of older mouse embryos, but are clearly disorganized in comparison with them (Hsu and Baskar, 1974). From observations of the tumors, the strongest evidence that the differentiated tissues are benign was obtained by Pierce et al., (1960). They found that approximately 36% of cystic embryoid bodies contained embryonal carcinoma cells. In the rest embryonal carcinoma cells had either undergone complete differentiation or necrosis. When single embryoid bodies were transplanted subcutaneously, only 31% gave rise to teratocarcinomas; the remainder formed well-differentiated benign tumors (that is, teratomas). The close correlation between the presence of embryonal carcinoma and the abili/Inner Prospective

ty of the embryoid bodies to form malignant teratocarcinomas suggests that the differentiated tissues of the tumors are not malignant. This idea can perhaps best be tested by growing differentiated teratocarcinoma cells in vitro, and examining their properties for signs of malignancy. The results to date are consistent with the idea that the differentiated cells of the tumors are benign (see below). That embryonal carcinoma cells are the stem cells of these tumors, and that a single embryonal carcinoma cell can give rise to a teratocarcinoma containing a wide variety of differentiated tissues, was first conclusively demonstrated by Kleinsmith and Pierce (1964) by intraperitoneal implantation of a single embryonal carcinoma cell taken from a simple embryoid body. This has since been confirmed by reinjection of clonal embryonal carcinoma cell lines grown in vitro (see below). Although all of the primary tumors, whether they are obtained spontaneously or are experimentally induced, are generally similar in composition, changes can occur when teratocarcinomas are retransplanted. These changes fall into three categories (Stevens, 1958). The first type of change is the loss of malignancy; teratocarcinomas can become benign because all of the embryonal carcinoma cells undergo differentiation or necrosis. The second type of change involves a restriction in the type of tissues found in the tumor: the differentiated tissues in such tumors are predominantly of one type, with embryonal carcinoma cells present. For example, a tumor which consists primarily of neural tissue and embryonal carcinoma cells is known as a “neuroteratoma.” Experiments ccl I mass

Tropho&last

Blastocoel

Moru

la

’

b.

a. Figure

Trophectoderm

4. Schematic

Representation

of Various

C. Stages

of Mouse

Embryogenesis

(A) Morula. The embryo consists of a ball of up to 64 cells. The outer cells will form the trophoblast, but are not yet functionally distinguishable from the inner cells, which will form the inner cell mass. (B) Blastocysts. The four-day old mouse embryo consists of an outer layer of trophectoderm, surrounding a fluid-filled cavity, and a group of pluripotent cells known as the inner cell mass. The dotted line shows where the next differentiation, the formation of the endoderm, will occur. (C) Embryo at approximately 5 days of development. The endoderm has formed on the free surface of the inner cell mass, and the two-layered embryonic structure is growing down into the blastocoel.

Teratocarcinomas 233

and Embryogenesis

with clonal cell lines grown in vitro suggest that such restriction of the differentiative capacity of embryonal carcinoma cells probably represents a change in the amount of each cell type formed, rather than a change in the range of tissues that they are capable of forming (Kahan and Ephrussi, 1970). In general, such “limited” tumors have relatively large amounts of embryonal carcinoma cells with decreasing ability to form complex differentiated tissues. The ultimate result of this type of regressive change is a tumor which consists of only embryonal carcinoma cells which do not give rise to any differentiated tissue (nullipotent embryonal carcinoma cells). The third type of change which may occur when teratocarcinomas are sequentially transplanted is the appearance of tumors which consist of only one type of differentiated tissue, with no embryonal carcinoma cells apparent. The most likely explanation for the origin of such tumors is that one of the differentiated benign tissues of the teratocarcinoma has undergone malignant transformation, has overgrown the other tissues of the tumor, and in subsequent passages is the only tissue present. The most common example of this type is the yolk sac carcinoma, which consists of endodermal cells which produce a large quantity of the mucopolysaccharide, Reichert’s membrane(Pierce, et al., 1962). Origin of Embryonal Carcinoma Cells For many years there have been two main theories of the origin of the stem cells of teratomas: that they arise from primordial germ cells, or that they originate from disorganized growths of early embryos. The evidence obtained from studies of both spontaneous and experimentally induced teratomas indicates that both of these theories are correct.

Figure

5. Cystic

Embryoid

Bodies

Histological sections of complex embryoid bodies, showing the fluid-filled cavity and a variety of embryonic tissues. Magnification: approximately X 80. Hematoxylin and eosin staining. The embryoid bodies shown here were formed in vitro, but are similar to those found in the ascitic fluid of animals bearing intraperitoneal teratocarcinomas.

Derivation from Primordial Germ Cells Although teratomas are extremely rare in laboratory mice, Stevens and Little (1954) discovered that approximately 1% of the male mice of inbred strain 129 had spontaneous testicular teratomas. Recently a subline of this strain has been developed in which 32% of the male mice have teratomas, most of which are benign (Stevens, 1973). By examining progressively younger mice, Stevens (1962) found that the spontaneous testicular tumors of 129 mice are apparent in the fetal genital ridge (that is, prospective testis) as early as the 15th day of gestation. These tumors apparently arise by proliferation of the primordial germ cells, which subsequently differentiate to form derivatives of all three primary germ layers. That the tumors arose from the primordial germ cells and not other cell types present in the fetal genital ridge was suggested by their ultrastructural similarities with embryonal carcinoma cells (Pierce and Beals, 1964; Pierce, Stevens, and Nakane, 1967) and by the high levels of alkaline phosphatase activity in these two cell types (Chiquoine, 1954; Damjanov, Solter, and Skreb, 1971 a). Another observation suggesting this origin was that teratoma tumors were rarely formed in strain 129 mice bred for a congenital absence of germ cells (homozygous for the Sl allele) (Stevens, 1967b). The study of these tumors was greatly facilitated by the discovery that the incidence of tumor formation could be increased by grafting genital ridges from embryos of approximately 12 days of age into the testes of adult isogenic recipients. Tumors which closely resembled the spontaneous teratomas could be obtained in approximately 75% of all grafts, compared with only a few percent in the unmanipulated gonads (Stevens, 1964, 1966, 1970a,b). No teratomas were ever obtained from primordial germ cells of female mice. Stevens (1968) points out that this difference between male and female primordial germ cells in their susceptibility to teratocarcinogenesis may be a consequence of the fact that in the male fetus the primordial germ cells remain diploid until after birth, while in the female they enter meiosis on the 13th day of gestation. Derivation from Embryos The idea that teratomas could develop from misplaced embryos is over a hundred years old, and numerous experiments to test this idea have been reported (reviewed by Damjanov and Solter, 1974a). Mouse embryos from 2 cell (1 day old) to the egg cylinder (7 day old) stages can give rise to teratomas or teratocarcinomas when transplanted to extra-uterine sites (Stevens, 1968, 1970~; Salter, Skreb, and Damjanov, 1970); when embryos younger than day 7 of development are grafted to ectopic sites, they undergo normal development in

Cell 234

situ to the day 7 stage before they become disorganized (Stevens, 1968). Embryos of day 8 or more of development cannot give rise to teratocarcinomas, but can form benign teratomas (Damjanov, Solter, and Skreb, 1971 b). In addition, teratocarcinomas are not produced unless the fetal portion of the embryo is included in the graft (Solter and Damjanov, 1973). These results suggest that it is the pluripotent cells of the day 7 embryo which give rise to embryonal carcinoma cells. In contrast to tumors derived from primordial germ cells, which are all male, either male or female embryos can give rise to teratomas or teratocarcinomas (Dunn and Stevens, 1970). While embryonal carcinoma cells can arise from primordial germ cells in only certain strains of mice (strains 129 and A/He), they can be obtained from extra-uterine embryos in many different strains (Damjanov and Solter, 197413). The kind of tumor produced (malignant compared with benign) is strain dependent, and may be influenced by the immunological response of the host (Damjanov and Solter, 1974b; Solter, Damjanov, and Koprowski, 1975). Embryo-derived teratomas can also be obtained spontaneously in the LT strain of mice (Stevens and Varnum, 1974). About half the females of this strain have ovarian teratomas, which apparently arise from disorganized growth of eggs that develop parthenogenetically within the ovarian follicles: all stages from dividing eggs, to germ-layer formation, to complex large teratomas are apparent. Most of the tumors are benign teratomas. Teratomas can also be obtained by ectopic implantation of embryos derived from eggs which have been parthenogenetically activated in vitro; some of the cells in such tumors may be haploid (lies et al., 1975). isolation In Vitro of Clonal Cell Lines from Teratocarcinomas Pluripotent Embryonal Carcinoma Cells The evidence that cells can remain pluripotent during growth in tissue culture has been demonstrated only recently. Clonal lines of embryonal carcinoma cells were first isolated in vitro from spontaneous testicular teratocarcinomas (Finch and Ephrussi, 1967; Kahan and Ephrussi, 1970; Rosenthal, Wishnow, and Sato, 1970), and later from teratocarcinomas derived from a day 3 embryo (Evans, 1972, Martin and Evans, 1974, 1975a,b) and from a day 6 embryo (Bernstine et al., 1973; Jakob et al., 1973; Jami and Ritz, 1974) of strain 129. The pluripotency of these cultures was demonstrated by injecting cells into mice and showing that they give rise to teratocarcinomas containing a variety of differentiated tissues. It could be argued that while the single cells from which the cultures originated were

pluripotent, during growth in vitro they lose their pluripotency and become committed to different developmental pathways, so that whereas the entire clonal population is pluripotent, the individual cells in it are not. Since most subclones of pluripotent clonal lines also can give rise to teratocarcinomas, however, it is clear that the pluripotency of any one cell can be maintained during its growth in vitro. Almost all pluripotent embryonal carcinoma cell lines so far described are tumorigenic in syngeneic mice; teratocarcinomas are formed within four weeks of injection of l-5 x 106 em bryonal carcinoma cells. Although a single cell will suffice in a small percentage of injections (Kleinsmith and Pierce, 1964), the results of Jakob et al. (1973) suggest that 2 x 104 cells are required for teratocarcinoma formation in the majority of animals inoculated. Nonpluripotent Embryonal Carcinoma Cells To study the nature of pluripotency, it would be useful to compare embryonal carcinoma cells which are pluripotent with those that are restricted in the range of cell types they can form. The work of Stevens (1958) has suggested that embryonal carcinoma cells can become restricted in their ability to differentiate in the course of transplantation from one animal to another. Experiments with clonal cell lines grown in vitro indicate that cells may lose the ability to differentiate after extensive periods of growth in culture. Kahan and Ephrussi (1970) thus found that while some cell lines show a decrease in the amount of various differentiated tissue formed in vivo, all clonal cell lines examined remained pluripotent over a great many generations in culture. There is as yet no conclusive evidence that embryonal carcinoma cells can become limited in their developmental potential so that they form only one type of tissue. Complete loss of pluripotence can occur in embryonal carcinoma cells. Such “nullipotent” cells have been described by Bernstine et al. (1973), who found that some pluripotent clonal embryonal carcinoma cell lines lost the ability to differentiate entirely during growth in vitro. Martin and Evans (1975a,b) have also described a clonal nullipotent cell line derived from a transplantable tumor which consisted of only embryonal carcinoma. Such cells have the morphological and biochemical characteristics of pluripotent embryonal carcinoma cells, but apparently they are incapable of differentiation in vivo or in Vitro. Differentiated Cells Embryonal carcinoma cells appear to IOSe their malignancy when they differentiate (Pierce et al., 1960). It should be possible to confirm this by isolating various differentiated cell types from teratocarcinomas and testing their ability to form tumors. Regardless of whether or not the somatic cell derivatives of embryonal carcinoma Cells are

Teratocarcinomas 235

and Embryogenesis

in fact nonmalignant, teratocarcinomas may prove to be an excellent source of cells at different stages of differentiation, some of which might otherwise be difficult to obtain from the animal. Clonal lines of differentiated cells derived from teratocarcinomas will also be useful for comparative studies with pluripotent embryonal carcinoma cells. Only a few clonal lines of somatic cells have as yet been derived from teratocarcinomas, and the data suggest that these cells are nontumorigenic. Clonal cultures of fibroblast-like cells have been obtained from the SIKR teratocarcinoma cell line (Evans, 1972; Martin and Evans, 1974); in contrast to embryonal carcinoma cells, these fibroblastic cells were found to be density inhibited in their growth and they did not form tumors in mice except after long periods. These cells, however, underwent malignant transformation in vitro, and subsequently they gave rise readily to fibrosarcomas when injected into mice. Several nontumorigenic somatic cell lines have also been derived from cultures of embryoid bodies: fibroblast-like and epithelioid cells (Bernstine et al., 1973), cardiac and skeletal myoblasts (Boon et al., 1974), and two nonclonal endodermal (parietal yolk sac) cell lines (Lehman et al., 1974). Characteristics of Embryonal Carcinoma Cells in Vitro Morphology and Growfh Although there are differences among clonal embryonal carcinoma cell lines, they all have certain features in common which can be taken as characteristic. In general, the cells are small (approximately 14 p diameter), rounded or slightly bipolar, which attach poorly to the substratum. They have relatively little cytoplasm, and the nucleus usually contains one or two large nucleoli. The cells adhere strongly to one another, and usually grow in colonies or “epithelioid nests”; in the phase contrast microscope the cell-cell boundaries are indistinct. Figure 6 shows a typical homogeneous culture of embryonal carcinoma cells. On the ultrastructural level, the cells are identical to those found in teratocarcinomas in vivo, with a cytoplasm almost devoid of organelles, except for a few mitochondria and a large number of free ribosomes. In general, embryonal carcinoma cell cultures are readily obtained from teratocarcinomas, providing that the cells are passaged on feeder layers of nondividing cells. The feeder-dependence of embryonal carcinoma cell growth, even at high density, was first noted by Kahan and Ephrussi (1970), and has been described by Martin and Evans (1975a,b). These observations offer an explanation for earlier results (Rosenthal et al., 1970; Evans, 1972; Martin and Evans, 1974), which showed that in the ab-

sence of added feeder cells, all pluripotent clones were heterogeneous, containing both embryonal carcinoma cells and fibroblast-like cells; it is likely that in those experiments, the fibroblastic cells substituted for feeder cells, and only heterogeneous clones were obtained because there was a strong selection against clones which were pure embryonal carcinoma. The origin of the fibroblastic cells in those cultures is unknown, but since differentiation of embryonal carcinoma cells occurs readily in vitro (see below), it is possible that the fibroblastic cells were differentiated derivatives of the embryonal carcinoma cells which arose in vitro at an early stage in the cloning procedure. Alternatively the “clones” may have in fact originated from two cells, one an embryonal carcinoma cell and the other a teratocarcinoma-derived fibroblast. The cloning method used (colony isolation) does not preclude the possibility that clones originated from two cells, particularly if such doublets were at a selective advantage. Homogeneous clonal embryonal carcinoma cell lines which are feeder independent have also been selected, although some of them require gelatin-coated surfaces for growth (Bernstine et al., 1973; Jakob et al., 1973; Jami and Ritz, 1974, and personal communication). Karyofype Most cell lines derived in vitro from mouse tumors, particularly those tumors which have been transplanted frequently from one animal to another, have abnormal sets of chromosomes (Terzi, 1974). It is therefore remarkable that almost all of the pluripotent embryonal carcinoma cell lines described have karyotypes similar to that of the normal mouse (40

Figure

6. Clonal

Embryonal

Carcinoma

Cell Culture

The individual cells are round or slightly bipolar, approximately 14 p in diameter, with a single large nucleolus. The cells are very adhesive to one another, and are almost always found in colonies, even when plated as a well-dispersed single cell suspension. Phase contrast microscopy. Magnification: X 180.

Cell 236

telocentric chromosomes). G&net et al. (1974) have studied the banding patterns of the chromosomes, and found that two of the pluripotent embryonal carcinoma cell lines that they examined had no detectable chromosomal abnormalities and thus appear to be perfectly euploid; one of these lines had been passaged in vitro for more than 300 generations. In contrast, the karyotypes of the somatic teratocarcinoma derivatives described above range from apparently normal to highly aneuploid. Although there is as yet no explanation for the exceptional chromosomal stability of pluripotent embryonal carcinoma cells, it is probably a function of their similarity to normal embryo cells: an understanding of the mechanism by which such cells can remain nearly euploid during growth in tissue culture may lead to an understanding of the maintenance of euploidy in the animal. G&net et al. (1974) found that their nullipotent embryonal carcinoma cell line had several chromosomal abnormalities, and have therefore suggested that a relatively normal karyotype may be required in order for cells to retain the ability to differentiate. On the other hand, Kahan and Ephrussi (1970) found that although most of their pluripotent clonal cell lines were nearly diploid, one line which was able to differentiate in vivo was nearly tetraploid. Studies of cell hybrids between teratocarcinoma cells and mouse fibroblasts indicate that the pluripotency of the teratocarcinoma cells is abolished and/or limited in the range of expression to that of the fibroblastic type (Finch and Ephrussi, 1967; Jami, Failly, and Ritz, 1973). These results are consistent with the idea that some “genie balance” is required for the expression of multipotentiality, but considerably more data are required before any definite conclusions can be drawn. Biochemical Properfies The only biochemical marker of teratocarcinoma cells which has been described in any detail is the enzyme alkaline phosphatase, which is present at high levels in embryonal carcinoma cells in vivo (Damjanov et al., 1971a). Bernstine et al. (1973) have found that clonal embryonal carcinoma cell lines express alkaline phosphatase activity during growth in vitro; although there is a wide variation in the specific activities of different embryonal carcinoma cell lines, even the minimal values are significantly higher than the specific activities of the somatic cell lines derived from the tumors. Nullipotent embryonal carcinoma cell lines have specific activities in the same range as the multipotent cell lines. Differentiation of Embryonal In Vitro It is clear from the preceding

Carcinoma

Cells

discussion

that many

aspects of embryogenesis can be studied using homogeneous populations of embryonal carcinoma cells. However, the potential value of teratocarcinomas as a model system for the study of embryogenesis can be realized only if embryonal carcinoma cells can differentiate in vitro. It has been possible to obtain differentiation of teratocarcinoma cells by explanting embryoid bodies, the ascitic form of the tumor (see above), and culturing them in vitro. On the basis of morphological and biochemical criteria, differentiation of the cells can be obtained in vitro providing two requirements are met. The first requirement is that the cells must be attached to a substratum. Using a tumor subline which produced almost exclusively the simple form of embryoid bodies, consisting of embryonal carcinoma cells surrounded by a layer of endodermal cells, Teresky et al. (1974) found that when the embryoid bodies were kept in suspension, even for periods up to six months, few new cell types were produced. In contrast, when the embryoid bodies were allowed to attach to the surface of a petri dish, a wide variety of cell types became apparent. Measurements of the levels of acetylcholinesterase (AChE) and creatine phosphokinase (CPK) activities (enzymes which are characteristic of several cell types, particularly muscle and nerve) also indicated that extensive differentiation did not occur unless the cells were allowed to attach to a substratum (Levine et al., 1974; Gearhart and Mintz, 1974). These results are consistent with the observation that in vivo, only a limited amount of differentiation occurs in suspension (in the ascitic fluid in the intraperitoneal cavity), while embryoid bodies attached to visceral, intraocular, or subcutaneous tissues show extensive differentiation (Pierce and Dixon, 1959a,b; Pierce et al., 1960; Stevens, 1960). A second requirement is that the cells must be grown without subculturing over a period of several weeks (Levine et al., 1974; Gearhart and Mintz, 1974). Teresky et al. (1974) observed that cells migrate out of simple embryoid bodies within 5-7 days after plating, and a great many new cell types are observed in the cultures during the next 30-120 days. The need for long term culture in order to obtain differentiation of teratocarcinoma cells has been emphasized by the work of Lehman et al. (1974). Using a nonclonal teratocarcinoma cell line, derived by subculturing embryoid bodies, they found that cultures which initially consisted of only two distinct cell types, embryonal carcinoma cells and endodermal (parietal yolk sac) cells, also differentiated over a period of 30-50 days into a variety of cell types, including pigmented cells, muscle, and cartilage. In contrast, the cultures could be maintained as relatively homogeneous embryonal

Teratocarcinomas 237

carcinoma days.

and Embryogenesis

cells

by subculturing

every

one or two

Differentiation of Clonal Cell Cultures While studies with explanted embryoid bodies and nonclonal lines derived from them have been useful in defining the conditions necessary for teratocarcinema cell differentiation in vitro, these studies have one major limitation: it is impossible to be certain that cell determination is occurring in vitro. It may be that some limitations of the developmental potential of the cells has already occurred in vivo, and that the differentiation observed is the terminal expression of previously determined cells. It is precisely this opportunity to study the processes of cell determination in mass cultures that is uniquely provided by teratocarcinoma cells. Although earlier attempts to obtain differentiation in vitro of clonal embryonal carcinoma cell cultures seemed to be unsuccessful, Jakob et al. (1973) noted that it did occur. A detailed description of the differentiation of clonal cultures derived from isolated single cells has been reported by our laboratory (Martin and Evans, 1975a,b; Evans and Martin, 1975). We found that the cells could be maintained as homogeneous embryonal carcinoma cultures providing they were subcultured every three or four days in the presence of nondividing feeder cells. When the cells were plated and maintained for several weeks in the absence of feeder cells, differentiation to a wide variety of cell types was observed. The most significant observation, however, was that the earliest stage in the differentiation of embryonal carcinoma cells in vitro is the formation of embryoid bodies which are identical to those found in animals bearing intraperitoneal teratocarcinomas. The pattern was always the same: pluripotent embryonal carcinoma cells plated as a single cell suspension in the absence of feeder cells initially formed small well-attached colonies. As the cells multiplied, these homogeneous aggregates became rounder and less well-attached to the substratum, and an outer layer of endodermal cells appeared (Figure 7A). Clonal nullipotent embryonal carcinoma cells cultured in the same way also formed aggregates, but no endodermal layer appeared (Figure 7B), nor did the cells show any other sign of differentiation in vitro or in vivo. Thus the first event of the differentiation of pluripotent embryonal carcinoma cells is the formation of endoderm. We reported the isolation of two types of clonal embryonal carcinoma cells, one which formed only simple embryoid bodies in vitro, which would undergo further differentiation only when attached to the substratum, and one which gave rise to simple embryoid bodies which rapidly became cystic when

kept in suspension (Figure 8). When simple or cystic embryoid bodies formed in vitro are allowed to attach to a substratum, endodermal cells migrate out and form a halo surrounding the aggregates within five days (Figure 9A). Over the next few weeks, the cultures rapidly become confluent, dense, and multilayered, and a variety of cell types become apparent, including cartilage, muscle, pigmented cells, and neural cells (Figure 9B). These results are summarized in Figure 10. These results were obtained with embryonal carcinoma cells derived from a tumor originating from a day 3 embryo. In fact, the formation of embryoid bodies in vitro had been observed in clonal cultures derived from a tumor originating from a day 6 em-

Figure 7. Aggregates of Clonal Embryonal Days after Plating a Single Cell Suspension. Magnification: (A) Pluripotent of endodermal (8) Nullipotent

Figure

8. Cystic

Carcinoma

X 200. Phase contrast microscopy. cells have formed embryoid bodies. cells is apparent. cells. No endodermal layer forms.

Embryoid

Body

Formed

Ceils

The outer

Five

layer

In Vitro

Four days after plating a single cell suspension of a clonal pluripotent embryonal carcinoma cell line, simple embryoid bodies are formed. If these are kept in suspension (in a bacteriological dish), they become cystic over the next 7 to IO days. The left side consists of a fluid-filled sac which may correspond to extra-embryonic yolk sac. The upper right side probably corresponds to the fetal portion of a day 8 embryo. These structures increase in size up to approximately 3 mm in diameter. Light microscopy of unfixed, unstained material. Magnification: X 90.

Cell 238

Figure

9. Differentiation

of Cells

in Embryoid

Bodies

Attached

to a Substratum

When simple embryoid bodies formed in vitro by a clonal pluripotent embryonal carcinoma cell line are allowed to attach to a tissue culture substratum, subsequent differentiation is extensive and a variety of cell types are formed. (A) Seven days after attachment, endodermal cells migrate out and form a halo around the embryoid body. Phase contrast.microscopy, Magnification: X 180. (B) Neural differentiation at the periphery of a halo of differentiated cells surrounding an embryoid body two weeks after attachment. Phase contrast microscopy. Magnification: X 110.

bryo (Jami and Ritz, 1974), and from a spontaneous tumor originating from primordial germ cells (Rosenthal et al., 1970). However, in these experiments embryoid body formation was not recognized as the primary event of differentiation since subsequent differentiation was poor, perhaps because these embryoid bodies were not allowed to attach to the substratum. It is not yet known whether the formation of embryoid bodies per se is a necessary step in the differentiation of embryonal carcinoma cells in vitro. If not, endoderm may nevertheless be the first cell type formed, although its appearance may not be readily apparent when the cells are not arranged in a three dimensional configuration. Alternatively, other cell types may be formed in the absence of endoderm formation. The early stages of differentiation in embryonal carcinoma cell cultures in vitro have certain features in common with the differentiation in the early mouse embryo. The endodermal layer of the embryoid bodies appears as the cell aggregates become less well-attached to the substratum (Martin and Evans, 1975), and this suggests that the cells on the outside of the clumps differentiate to endoderm while the inner cells remain unchanged. This mechanism is similar to that proposed for the early events of embryogenesis in the mouse (Tarkowski and Wroblewska, 1967). The first determination which occurs in the mouse embryo is the formation of the trophectoderm. There is now good evidence that the cells which become committed to form trophectoderm do so as a result of their position on the outer surface of the morula (Figure 4A; re-

viewed by Herbert and Graham, 1974). In the development of the mouse embryo, the second cell type to appear after the establishment of the blastocyst is the endoderm, which forms only on the free surface of the inner cell mass (Figure 48). The stimulus for the formation of endoderm may also be a positional one, as it has been found that inner cell masses isolated from the embryo form endoderm over the whole of their outer surfaces (Rossant, 1975; Gardner and Papaioannou, 1975). The first event of differentiation of pluripotent teratocarcinoma cells in vitro is thus similar to that of the inner cell mass of the mouse embryo. This, taken together with the observation that the transition from simple to cystic embryoid bodies closely parallels the subsequent differentiation in the mouse embryo (Martin and Evans, unpublished data), suggests that studies of the differentiation of embryonal carcinoma cells will be useful in understanding the process of normal embryonic cell differentiation. Immunological Properties of Embryonal Carcinoma Cells Since embryonal carcinoma cells can be derived from early embryos and also retain their most characteristic property, the ability to differentiate into a wide variety of tissues, it is reasonable to assume that they might also have antigenic properties in common with early embryo cells. For example, the major histocompatibility antigens of adult mice H-2 should be absent from embryonal carcinoma ceils, as they are from early embryos (reviewed by Edidin,

Teratocarcinomas 239

and Embryogenesis

a

b

C

Figure 10. Schematic bryonal Carcinoma

Representation of the Behavior Cell Cultures In Vitro

of Clonal

Em-

(a) Nullipotent Cells. After plating on a tissue culture surface in the absence of feeder cells, these embryonal carcinoma cells form flat homogeneous colonies. During the next few days these colonies become rounder and less well-attached to the substratum. They can remain attached or they can be detached and kept in suspension. Although cells migrate out from attached colonies, these are still embryonal carcinoma cells. Under no circumstances does differentiation occur in these cultures. (b) Pluripotent Cells which Form only Simple Embryoid Bodies. After plating on a tissue culture surface in the absence of feeder cells, these embryonal carcinoma cells form homogeneous colonies. During the next few days these colonies become rounder and less well-attached to the substratum. At this time an endodermal layer becomes apparent on the outer surface of the colonies. These “embryoid bodies” can be detached from the substratum and kept in suspension, where no further differentiation occurs, If, however, they are not detached from the substratum, or if the detached ones are allowed to reattach, the endodermal cells migrate out, and during the next few weeks the cultures become complex, multilayered, and a variety of new cell types appear. (c) Pluripotent Cells which Form Cystic Embryoid Bodies. These cells behave in the same way as those described above(b), except that cell differentiation continues in suspension, and the simple embryoid bodies that initially form, rapidly become cystic. If these embryoid bodies remain attached, or are allowed to reattach to the substratum, endodermal cells migrate out, and with time the cultures become dense, multilayered, and an even greater variety of cell types appear as compared with the cultures from embryoid bodies that remain simple if kept in suspension (b).

Gooding, and Johnson, 1974). Present evidence suggests that H-2 antigens are not present on the surface of embryonal carcinoma cells (Artzt and Jacob, 1974; Edidin et al., 1974). Positive antigenic similarities between embryonal carcinoma cells and early embryo cells were first detected by Edidin et al. (1971; Gooding and Edidin, 1974). They immunized rabbits with cells derived from embryoid bodies of a mouse teratocarcinoma and obtained an antiserum which reacted against teratocarcinoma cells and also against early mouse embryos. The serum, however, contained at least three distinguishable antibody specificities, including activity against several other cell types such as polyoma and SV-40-transformed mouse fibroblast lines and a variety of mouse tumor cells. A more specific antiserum was obtained by Artzt et al. (1973) by immunizing syngeneic mice with clonal nullipotent embryonal carcinoma cells. The results of cytotoxicity and indirect peroxidase tests indicated that this antiserum was active against embryonal carcinoma cells, mouse sperm, and early mouse embryos, but had no activity against somatic cells derived from a teratocarcinoma or other nonteratocarcinoma cell types tested, including SV-40 and polyoma-transformed ceils. The expression on early embryo cells of the antigen(s) recognized by this serum was found to increase from little or none on fertilized one cell eggs, to a maximum on the 8 cell stage. Recently, we have obtained an apparently similar antibody to that described by Artzt et al. (1973), raised in syngeneic mice against a pluripotent embryonal carcinoma cell line, that stains ceils of the 16-32 cell stage embryo by the indirect immunofluorescence test (Stern, Martin, and Evans, unpublished data). These results therefore indicate that embryonal carcinoma cells, but not their differentiated derivatives, share specific antigens in common with early mouse embryos and sperm. T Locus Expression A series of mutations which are important in normal mouse embryogenesis has been defined at the T locus complex. They affect morphogenesis in the mouse in very specific ways: for example, the t’2 mutants affect blastocyst formation, the to mutations affect the division of the inner cell mass into embryonic, and extraembryonic ectoderm, the tw’ mutations affect growth and maintenance of the neural tube and brain, and so forth (reviewed by Gluecksohn-Waelsch and Erickson, 1970). Most important, the T locus genes have been shown to specify antigens expressed on the surface of sperm (Bennett et al., 1972; Yanagisawa et al., 1974). Artzt, Bennett, and Jacob (1974) therefore reasoned that a gene at the T locus might specify the antigen(s) common to sperm, early embryos and

Cell 240

embryonal carcinoma cells. They focussed their attention on the gene t’*, which in the homozygous state acts earliest and blocks development at the morula stage. They tried to prepare an anti-sperm antibody specific for the product of the wild-type allele of the t+2 gene (+ ++2), and show that this serum reacted with embryonal carcinoma cells. They found, however, that for a variety of reasons it is not possible to prepare anti-sperm antibodies specific for the products of the wild-type alleles of the t genes of the T locus, although antibodies specific for the products of the mutant T locus alleles have been obtained. To determine whether embryonal carcinoma cells carry antigens specified by the wild-type allele (++I*), Artzt et al. (1974) were therefore obliged to perform quantitative absorptions of anti-embryonal carcinoma serum with sperm from wild-type animals compared with sperm of animals heterozygous for the ++I2 allele, and then tested the absorbed sera for cytotoxicity against embryonal carcinoma cells. Since sperm cells appear to express each allele carried by the animal, for any given reduction in cytotoxicity the serum would have to be absorbed with twice as many sperm from the heterozygous t’2/ + +I* animal as for the wild-type + ++2/ + +I2 animals. (Theoretically, absorption with sperm from a homozygous t+2/t+2 animal should not remove any antibody activity against embryonal carcinoma cells. Since this gene combination is lethal in early embryogenesis, no such sperm could be obtained to perform these controls). The results indicated that, as expected, twice as many sperm from the heterozygous animal were required to reduce the cytotoxicity of the serum to 50% of its initial activity against embryonal carcinoma cells. Absorption of the serum with sperm from animals bearing other mutations in the T locus gave the same results as absorption with sperm from wild-type animals. This suggests that the major antigen on embryonal carcinoma cells is the product of the wild-type allele of the tl2 gene (+ +12), and that by analogy with previous results (Artzt et al., 1973) antigens specified by + +I2 are also present on normal embryos of the 2-8 cell (and later) stages. This latter conclusion has yet to be confirmed by demonstrating that absorption of antiembryonal carcinoma cell serum with ++I2 sperm removes activity of the serum against early embryo cells. All of these results are consistent with the hypothesis that each stage of embryogenesis is triggered by the appearance of specific genetically controlled cell-surface components, which function as recognition sites (Bennett, Boyse, and Old, 1971). However, antigens specified by T locus genes have not yet been demonstrated on the surface of early embryo cells except for antigen(s) apparently specified

by the + +I2 allele. These have been detected using antiserum raised against embryonal carcinoma cells, If the antigen(s) recognized by the anti-embryonal carcinoma cell serum is indeed important in development of the embryo, strong selective constraints might have conserved it through mammalian evolution. It is therefore interesting that antiserum against mouse embryonal carcinoma cells recognizes antigen(s) on human sperm (But-Caron et al., 1974), and also on the sperm cells of bull, but not of rooster (Fellous et al., 1974). On human and mouse sperm cells, this antigen is apparently located in the postacrosomial region of the sperm (Fellous et al., 1974). But-Caron and her co-workers point out that the presence of such antigen(s) on human sperm suggests that genetic loci similar to the T locus of mice may also exist in man (and other mammals). Embryonal Carcinoma Cells as Normal Embryo Cells It is clear that numerous similarities exist between the cells of the early embryo and embryonal carcinoma cells, the stem cells of teratocarcinomas. Pluriporency-the ability of both cell types to proliferate in the undifferentiated state and at the same time to give rise to differentiated somatic cell types. Ultrastructure-both cell types have the morphology of the undifferentiated cell type. Biochemical properties-alkaline phosphatase activity is present at high levels in both cell types. Surface properfies-the two cell types have at least one specific surface antigen in common, which has so far not been found on any other cell type except sperm. Formation of embryoid bodies in vifro-differentiation of embryonal carcinoma cells occurs by a process similar to normal embryogenesis. The fact that endoderm is the first differentiated cell type formed by embryonal carcinoma cells in vitro suggests that on a functional level all embryonal carcinoma cells most closely resemble the cells of the inner cell mass of the day 4 embryo. No significant differences have yet been detected among embryonal carcinoma cells found in tumors from different sources (that is, spontaneous tumors derived from primordial germ cells compared with those derived spontaneously or experimentally from embryos of different ages, up to 7% days). There are at least two possible ways in which embryonal carcinoma cells could arise from their progenitor cell types. First, some malignant change occurs in the progenitor cell types. Second, that embryonal carcinoma cells are normal undifferentiated embryonic cells (or primordial germ cells) which behave abnormally because they are not in their normal environment. This idea, (discussed by Damjanov and

Teratocarcinomas 241

and Embryogenesis

Solter, 1974a), is perhaps not as iconoclastic as it may seem: at the end of the 19th century, Cohnheim and Ribbert argued that tumors arose from embryonic cells released from the normal restraints imposed by surrounding tissues (see Willis, 1967). The concept that embryonal carcinoma cells are normal pluripotent embryo cells is suggested first of all by the observation that they can be obtained with high frequency from early embryos transferred to extra-uterine sites (Damjanov and Solter, 1974b). Thus if some malignant change does occur, it is one which occurs very readily. The second suggestive point is the remarkable chromosomal stability of embryonal carcinoma cells over a very large number of generations in vivo and in vitro. No other mouse tumor cell type has ever been reported to remain euploid in this way. Also significant is the fact that no differences have as yet been detected between embryonal carcinoma cells and the cells of the early embryo. In particular, at least one characteristic of embryonal carcinoma cells, their agglutinability with Concanavalin A (Oppenheimer and Odencrantz, 1972), which is considered typical of malignant cells, is also characteristic of early mouse embryo cells (Pienkowski, 1974). If it were true that embryonal carcinoma cells are normal pluripotent embryo cells, and if it were possible to obtain pluripotent embryo cells by culturing early embryos in vitro, then such cultures should have the same in vitro characteristics as embryonal carcinoma cell cultures, and should form teratocarcinemas when injected into mice. Although several cell lines have been obtained from early mouse embryos in vitro (Sherman, 1975), all of those isolated so far are of a somatic cell type and are neither pluripotent nor similar to embryonal carcinoma cells. Perhaps the most critical test of the idea that embryonal carcinoma cells are normal embryo cells would be to determine whether or not, given the “correct” environment, they can form a normal fertile animal. The most appropriate environment to place them in would probably be the inner cell mass (ICM) of a day 4 blastocyst (Figure 48). Such experiments can be done, as Gardner (1968) has shown that cells from the ICM of a donor embryo can be inserted into the ICM of a recipient blastocyst. Such a “chimeric” embryo can be placed in a suitable foster mother, grown to term, and mice showing characteristics of both donor and recipient embryos are born. Brinster (1974) has already reported experiments in which he transferred teratocarcinoma cells from dissociated embryoid bodies into recipient blastocysts from Swiss Albino mice and then placed these blastocysts into foster mothers. Of the sixty mice born, one had patches of pigmented hair, which could only have come from the donor terato-

carcinoma cells. While these results are preliminary, and experiments must be performed using criteria for chimerism other than coat color (for example, isozyme markers), it seems possible that embryonal carcinoma cells can participate in the formation of a normal mouse. Acknowledgments I would like to thank Dr. Robin Weiss for introducing me to the subject of teratocarcinomas, Dr. Martin Evans for the opportunity to work in his laboratory, and Dr. G. Steven Martin for helpful suggestions during the preparation of this manuscript. Figures 3, 5, 6, 7, and 9 are reproduced from Martin and Evans (1975). The original research described here was supported by a grant from the Cancer Research Campaign. I am presently supported by a fellowship from the US Public Health Service National Cancer Institute.

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