Cell Lines Derived from Teratocarcinomas1tal teratocancinomas, or, like line LT,, from ovarian terato carcinomas (H. Jakob, unpublished results). In contrast, multipotential EC cells

(CANCER RESEARCH 36, 4224-4231 , November 1976J

Summary

A variety of cell lines have been isolated in vitro fromtransplantable teratocarcinomas. Some of them correspondto embryonal carcinoma (EC) cells. They are malignant andrepresent the stem cells from which all differentiated tissuesderive in the tumor on in vitro. EC cells share some biochemical and antigenic properties with multipotential embryonic cells.

From one of these EC cell lines, variants have been isolated in vivo and in vitro. Some are of EC type but restrictedin their pattern of differentiation; others are altered in theirtumonigenicity on in their antigenic characteristics. Anotherclass of such variants corresponds to non-EC types. Themost interesting ones correspond to tumonal lines of extraembryonic tissues.

All these cell lines constitute a valuable material for thestudy of mouse development and differentiation.

Introduction

Tenatomas were first observed in mice by Stevens (43) andhave been studied in detail by Stevens (39) and Pierce (34)[for review, see the paper of Oamjanov and Solten (12)].Testicular tenatomas occur spontaneously in strain 129mice and appear to be derived from primitive germ cells(40). Ovarian teratomas are found in LT mice and seem toresult from the high incidence of spontaneous parthenogenotes occurring in this strain (44). Tenatomas can, inaddition, be induced with high efficiency in a variety ofinbred mouse strains by grafting either early embryos ongenital ridges into the testes (41, 42). Whether spontaneousor induced, some of these tenatomas can be serially transplanted in syngeneic adult mice. In contrast to other tumons, teratomas contain a wide variety of tissues comesponding to derivatives of the 3 germ layers. When transplantable, they contain, in addition, embryonic-like cellscalled EC cells.3 These EC cells exhibit 3 important properties: (a) they are malignant; (b) they can differentiate intoderivatives of all 3 germ layers and even be â€˜â€˜redirectedâ€•andparticipate to the production of normal mice when injectedinto a blastocyst (10, 19, 29, 32); (c) their derivatives aregenerally nonmalignant (39).

, Presented at the Conference, â€˜â€˜Regulation of Gene Expression in Devel

opment and Neoplasia,â€•June 2 to 5, 1976, Santa Ynez, Calif. This work wassupported by grants from the NIH (Grant 1 R 01.CA.16355.01), the AndrÃ©Meyer Foundation, the Centre National de Ia Recherche Scientifique (LA 88),the DÃ©lÃ©gationGÃ©nÃ©ralea Ia Recherche Scientifique et Technique(73.7.1208), and the Fondation del Duca.

2 Presenter. To whom correspondence should be addressed.

3 The abbreviation used is: EC cells, embryonal carcinoma cells.

In the past few years, mouse teratomas have arousedconsiderable interest because they provide a material suitable for the study of differentiation on of certain aspects ofcancer either in the mouse embryo on in vitro.

In this paper, we shall discuss the isolation and cultivation of EC cells and their properties, their in vitro differentiation, and the derivatives that can be obtained from them,both in vivo on in vitro.

Isolation of Cell Lines from Teratocarcinoma Tumors

Teratocarcinoma cells can be obtained in culture eitherfrom solid tumors or from the so-called embryoid bodiesfound in the ascites formed after i.p. injection of tumorfragments. Two types of embryoid bodies can be distinguished: (a) a cystic form composed of a single layer ofpanietal yolk sac-like cells arranged around a fluid-filledcavity, and (b) a solid form composed of a layer of endodermal cells surrounding a core of EC cells. When put inculture, both fragments of solid tumors containing EC cellsand embryoid bodies grow and differentiate into a variety ofcell types. From such cultures, cell lines can be isolated andestablished in vitro.

On the one hand, a series of differentiated cell lines, suchas myocard (PCDI), myoblast (PCD2) (8), and neuroepithelial cell lines, have been isolated (25). Like similar celltypes established in culture from mice, most of these differentiated lines are aneuploid. Although obtained from tenatocarcinomas, they are generally not tumonigenic and behave like similar cells isolated directly from embryonic onadult mice. For instance, the teratoma-denived myoblastline fuses and produces myotubes in culture under conditions similar to those used with either primary cultures ofmouse myoblasts on a cell line derived from a rat embryo (8,11).

A number of EC cell lines have also been established invitro (7, 16, 21, 22, 25, 28, 31, 41). Upon injection intosyngeneic mice, all these cell lines give tumors, but 2classes have been distinguished according to the cell typespresent in the tumors (7). Nullipotential lines are supposedto give rise to tumors containing almost exclusively ECcells. This is the case for F9, which has been extensivelyused for immunological studies (3). Both in vivo and inculture, it produces EC cells. These are accompaniedsometimes by a few primitive endoderm-like cells. Similarlines have been isolated either from testicular or experimental teratocancinomas, or, like line LT,, from ovarian teratocarcinomas (H. Jakob, unpublished results). In contrast,multipotential EC cells give rise to tumors containing alarge variety of differentiated cell types. It has been repeat

4224 CANCERRESEARCHVOL. 36

Cell Lines Derived from Teratocarcinomas1

J. F. Nicolas, P. Avner, J. Gaillard, J. L. Guenet, H. Jakob,2and F. JacobService de GÃ©nÃ©tiqueCellulaire de l'Institut Pasteur et du College de France, 25 rue du Dr. Roux, 75015 Paris, France

on March 30, 2020. © 1976 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

Ce!! Lines Derived from Teratocarcinomas

edly and unambiguously demonstrated both by injection invivo of single cells from embryoid bodies (23) and by in vitrodifferentiation of EC clones (31) that single cells can produce derivatives of all 3 embryonic germ layers. Multipotential EC cell lines are thus able to differentiate in vivo, and itseems likely that most of these lines are also capable ofdifferentiating under appropriate conditions in culture (25,28, 31, 36, 38).

A series of EC cell lines had thus been isolated from thetransplantable teratocancinoma 0TT6050 which was obtamed by On. L. Stevens from a graft of a 6-day 129 embryointo the testis of a 129 mouse. In the remainder of thispaper, we shall concentrate on some of these lines, especially PCC3, which differentiates easily in culture.

Properties of EC Cells

EC cells are small epithelial cells that have indistinct cellboundaries and that contain characteristic nefningent granules of a yet unknown nature. Their nucleocytoplasmic ratiois high. Their cytoplasm, as seen in electron microscopy,contains few onganelles, some mitochondnia, a large numben of polysomes, and no endoplasmic reticulum.

Cultural Conditions

In our laboratory, EC cells are grown in Oulbecco's mod ified Eagle's medium supplemented with 15% fetal calf serum. These cells are particularly fragile and lyse easily.Some laboratories have found it convenient to cultivatethem on feeder layers of other differentiated cell types, suchas mouse or rat fibroblasts (28, 38), or on gelatin-coatedplates. For the sake of simplicity, our laboratory has decided to cultivate these cells directly on the plastic surfaceof cell culture plates. The cultures are maintained in ahumid air-12% CO2 atmosphere. The cells are plated at arather high density (104/sq cm) and neplated 48 hr afterdissociation by pipetting. Under such conditions, the cellsremain in exponential phase and do not differentiate inculture (21). The efficiency of cloning ranges from 20 to80%, depending on the line.

Tumorigenicity and Mu!tipotentia!ity

Tumors can be produced by s.c. or i.p. injection intosyngeneic mice. For the F9 line, 5 x 102 cells injected s.c.give tumors in 100% of the animals, but the number of cellsdepends on the strain. With PCC3, more than 10@cells arerequired. In the testis, which is considered to be an immunologically privileged site, 10 PCC3 cells or less are sufficient to produce tumors in 100% of the injected animals.

The tumonigenicity of nullipotential lines such as F9 onLT, appears to be very stable, as do both the tumonigenicand multipotential characters of other established lines. Forinstance, a total of 20 clones have been isolated oven 5everal years from PCC3. All these clones have given tumorscontaining derivatives of the 3 germ layers. Similar observations of stability have been made with another line, PCC4,

and during the isolation of drug-resistant variants (see below).

Cytogenetics

In contrast to the majority of other mouse cell lines established in culture, whether malignant on not, most of the EClines exhibit a narrow karyotypic distribution with a modeequal or close to 40, the normal mouse chromosome complement. The 1st examination of the banding pattern observed in the EC lines isolated in our laboratory led to theconclusion that some of these lines were perfectly euploid(18). A more refined study carried out in collaboration withOr. L. Evans has, however, invalidated this conclusion. Allour lines, derived from tumor OTT6O5O,contain some chromosomal rearrangements. In all cases, the Y chromosomeis absent, as it is absent from an old stock of 0TT6050embmyoid bodies received from On. Stevens and frozen in1971 by Or. B. Ephrussi. To our knowledge, no perfectlyeuploid EC line has been described up until now.

Strain PCC3 (Fig. 1) has a deletion of Chromosome 14(this at first sight appeared to mimic a Y) and a tnisomy 19.This karyotype appears to have remained extremely stablein culture over more than 1000 generations. There does notseem to exist any particular anomaly which, occurring in allEC lines, can be associated with the EC condition; theselines exhibit slight but different changes. PCC4 has nodeletion of Chromosome 14 but has 2 isometacentnic chromosomes (Nos. 1 and 13); PCC1 has neither deletions norisometacentnics but has a nonidentified small sumnumerarychromosome. Since all these strains derive from the sameoriginal tumor, these alterations must have occurred duringcellulargrowth,eitherin vivoduringserialtransferon invitro during serial passage.

Biochemical Markers

A number of enzyme activities known to vary during embryonic development have been studied as biochemicalmarkers. High alkaline phosphatase activity has been associated with EC cells (7), and it has been confirmed byhistochemical techniques that both the nullipotent strain F9and the multipotential strains PCC3 and PCC4 have highactivity. No marked acid phosphatase activity is present,and f3-glucuronidase activity is very weak in all 3 strains.During the in vitro differentiation of PCC3/A/1 (see below),alkaline phosphatase activity disappears very quickly frommost of the cells, while acid phosphatase increases markedly (Table 1). fJ-Glucuronidase activity (as measured histochemically) increases, but in only a minor fraction of the cellpopulation.

a-Fetoprotein is known to be secreted by the fetal yolksac and liver (1). It is also present in some teratocancinomasand hepatomas (1, 15, 45). EC cells of both nullipotential(F9) and multipotential strains (PCC3 and PCC4) have beenshown by immunochemical techniques not to secrete it.This correlates with the results of Mintz, who noted itsabsence from the comes of embryoid bodies (30). While

NOVEMBER1976 4225



Characteristicsof Ce!IstrainsHistochemicaltestSerologyAv.

no. of(%

positive cells)(% positive cellsexpressing)Alkalinechromo

phospha- Acid phos F9 anti- PCC4 H-2antiType of cellsStrainTumor typesomestase phatasegen antigengensECF9EC

only4180 NTa9(JbOrECPCC4Trigerminal

teratocarcinoma3970

050b 9@r041ECâ€•PCC3/A/1Trigerminal

teratocarcinoma40100

050r90rECePCC3/S640Ectoderm

and endoderm only40NT

NT50â€• NTNTECPCC3/A/1/O-G3'Trigerminal

teratocarcinoma39-40100

NT60r 70rNTFibroblasticPCC3/A/1/O-G1'Trigerminal

terato 65-700 0Or 9@r0@like@carcinomaEpithelialPCC3/A/1/O-G2'Trigerminal

teratocarcinoma70-750

NTOrorHeteroge

Differentiation at402-5 1007'@0â€•neousconfluency;bulknon-EC.

cells, 28daysMyocardialPCO160-700

1000 O@+â€œMyoblasticPCD260-7050-701000O'@EndodermalPYS

C3Parietal yolk saccarcinomaNTNT

NTNT NTNTCytotropho

T.O.M.1Trophoblastoma65-750 NTNT NTNTblastic

J. F. Nicolas et a!.

Table 1

S NT, not tested.

@ Cytotoxicity test.r Immunofluorescence test.

41 Tested by absorption.

e Strains able to differentiate in vitro.

I PCC3/A/1/O-G1 , -2, and -3 are differentiated on media without glucose (â€”G); -1 and -2 with galactose present, -3 with pyruvate

present.

tumors of nullipotential line F9 equally do not contain a-fetoprotein, both the solid tumors and the embryoid bodiesformed by the multipotential strain PCC4 do; it has alsobeen found in in vitro-differentiated aggregates of PCC3/A/1. The secretion of a-fetoprotein thus characterizes thepresence of certain early differentiated derivatives of ECcells. @Thesecould well correspond to distal endodermalcells, since neither panietal yolk sac carcinomas, the PYS-2cell line (25), nor the trophoblastoma (see below) secretesa-fetoprotein.

AntigenicMarkers

The group of major histocompatibility H-2 antigens cannot be detected at the surface of EC cells (4, 14, 31, 46). Inrecent years, immunization of either mice or rabbits witheither teratocarcinoma cells in culture or embryoid bodieshas led to the description of several antigens (20). Sinceneither the cells used for immunization or as targets nor thesera obtained have been compared from one laboratory toanother, we will mention only those antigens described inour laboratory.

The F9 antigen was detected with a serum obtained byhypenimmunization of male syngeneic 129 mice with Xirradiated nullipotent F9 cells in culture. It is absent from allthe adult tissues examined, including kidney and brain, thesole exception to this being the male germ line (3, 17) (the

female germ line has yet to be investigated). The F9 antigencan be detected on the surface of the egg after fertilization;it increases in amount up to the morula stage; it is presentboth on the trophectoderm and the inner cell mass of theblastocyst and can still be detected on the embryonic ectoderm up to Day 9 (M. H. Buc, unpublished data). It is genetically determined by the wild haplotype of the T locus andbehaves in crosses as allelic to the t'2 haplotype (2). The F9antigen is present at the surface of all EC cells examined sofar, with one exception to be described below.

Another antigen, PCC4, has recently been found on thesurface of EC cells. It was detected with a serum obtainedby hypenimmunizing syngeneic 129 mice with X-irradiatedmultipotential PCC4 cells in culture. After absorption withF9, PYS-2, and PCDI myocard cells, the serum still exhibits an activity against PCC4 cells (Table 1). This activity isnot detectable on adult tissues other than sperm. It is notpresent on either morulae or the trophectoderm, but appears on the inner cell mass. The PCC4 antigen has beendetected on the surface of all multipotential EC cells exammed so far (G. Gachelin and A. Kemler, unpublished data).

EC Cells and Wruses

The eventual production of viral particles was investigated in several EC cells including PCC3. This investigationwas carried out either on exponentially growing cells or on





cells previously treated to a brief exposure of bromodeoxyunidine and dexamethasone. A variety of tests, such as theaddition of EC cell culture medium to a variety of cell typesin culture, the injection of newborn mice, the incorporationof radioactive unidine, and examination of cell sections byelectron microscopy, failed to detect the presence of viralparticles in these cells. This result is interesting when cornpared with observations made for teratoma derivatives suchas panietal yolk sac carcinomas which, in the electron microscope, appear to be filled with intracisternal type A viralparticles (35). In contrast to this, most cells differentiated invitro for 4 weeks are devoid of this type of particle. It is asthough a provirus, present in the genome, becomes activated only when a certain program(s) of differentiation isput into operation.

The small DNA viruses, such as polyorna and SV4O, canneither grow in EC cells nor transform them. AlthoughSV4O, for instance, can enter the cell and lose its capsid(24), it is unable to multiply or to express any of its earlyfunctions. Viral multiplication can occur only in those teratoma-denived cells which have reached a more advancedstage of differentiation.

Certain small RNA viruses, such as mengovirus, appear tomultiply normally in EC cells (24) as well as larger DNAviruses such as adenovirus.4

In Wtro Differentiation

EC cells remain in an undifferentiated state when maintamed in exponential growth phase by frequent replating.Otherwise, the cells of strain PCC3 begin to differentiate.This differentiation can be followed either in aggregates orafter confluency in culture dishes.

In Aggregates.EC cellsadhereverystrongly,one toanother, and it is easy to get aggregates of cells out of asuspension either by centnifugation or by leaving the cellsunder such conditions that they cannot attach to the Petnidishes (27, 31).

When centrifuged aggregates are replated in tissue cultune dishes, differentiated types of cells appear very rapidly.The 1st morphologically distinguishable types correspondto very flat epithelial-like cells with numerous cytoplasmicprojections. These cells can migrate and do not adhereclosely to each other. They always appear at the peripheryof the clones where they form a crown . The 2nd rnorphologically differentiated type is observed between these epithehal cells and the undifferentiated ones. They are fibroblastlike and are in close contact with each other. On this layer ofdifferentiated cells, nervous-type cells appear later on.These 2 early differentiated cell types are, unlike EC cells,alkaline phosphatase negative and do not possess antigensrecognized by an anti-F9 serum. Under these conditions,cells with an endoderm morphology are rarely seen. Incontrast, if such aggregates are left in suspension for several days, a certain proportion of them form an externallayer of endodermal type cells, the final structure resernbling embryoid bodies. However, when such aggregates areplated out early, endodermal cells are rarely seen prior to

4 Kelly, F., and Boccara, M. Susceptibility of Teratocarcinoma Cells to

Adenovirus Type 2. Nature, 262: 409-410, 1976.

the formation of the other 2 differentiated cell types. Later,aggregates in suspension form different types of tissues,similar to those found in the confluent differentiation systern. In both systems, the formation of the first differentiatedcell types occurs only after the aggregates reach a certaincritical size. The larger the aggregate, the more quicklydifferentiation occurs.

Differentiation after Confluency. When PCC3 or itsclones are maintained in culture without replating, manytypes of differentiated cells appear as a function of time.These different forms can be seen directly in the culture onin histological section after fixation of the multilayered cellsheath (Fig. 2).

Three or 4 days after plating, the culture reaches confluency. Triangular flattened epithelial forms then appearwhich are highly contact inhibited. These cells can eitherform spherical vesicles that frequently detach from the dish,or they can transform into large cells containing severalnuclei. They closely resemble endodermal cells. After ashort period of partial lysis, areas of nervous tissue appearbetween Days 12 and 15. When the multilayer is dissociatedprior to the 12th day, most of the cells resemble the 1st 2cell types appearing in aggregates. By Day 7, only 3% of thecells are residual EC cells. Nervous differentiation is in turnfollowed by the appearance of unicellular contractile cells,probably myocard. Around Day 22 to 24, cartilage appears, and this is followed by the appearance of lipid cells,zones of keratin, areas of contractile skeletal muscle, andpigmented retinal epithelial cells. This pattern of in Vitrodifferentiation is highly reproducible. It is similar to thatdescribed by Martin and Evans (28) for an EC line derivedfrom another transplantable teratocarcinoma, OTT 5568.

The distribution of tissues observed depends on the cultune conditions, especially the batch of fetal calf serumused. The presence of glucose also appears critically necessary for the differentiation sequence to reach completion.In the absence of glucose, differentiation is apparentlyblocked at some time between Days 11 and 22 of the differentiation sequence (5).

The differentiated layers of cells can be dissociated withtrypsin, and certain of their properties can be examined.From these studies, the following conclusions can bedrawn: (a) The bulk of the cells appear to have the samekanyotype as the original PCC3, a result that indicates thatdifferentiation can occur in vitro without major change inkaryotype. (b) The bulk of these cells are no longer tumonigenic. When injected in the testis, more than 10@cells arenecessary to obtain a tumor, whereas less than 10 cells aresufficient for the original PCC3. (C) They lose the F9 antigenand acquire H-2 (31).

Teratocarcinoma Derivatives Obtained in Vivo

Ascitic tumors containing embryoid bodies can be obtamed by injection of fragments of solid testicular tumorsinto the penitoneal cavity of syngeneic mice. Undistinguishable ascitic tumors with embryoid bodies can also be obtamed by injecting multipotential EC cells such as PCC3. Allthese tumors can be serially transplanted and, in most

4227NOVEMBER1976



J. F. Nico!as et a!.

vitro which affect one or another original property of thecells.

Drug-resistant Variants. Clones resistant to azaguanine(30 @g/ml)and that are devoid of hypoxanthine-phosphonibosyltransfenase activity (Hprt) (26), as well as ouabainresistant (3 mM) (6) and bromodeoxyunidine-resistantclones (TK@)(13), have been obtained from several EC lines.Such resistant cells can be obtained without any noticeablechange in their karyological, tumonigenic, or in vitro differentiation properties.

Carbon Source Variants. As previously mentioned, differentiation is blocked in the absence of glucose. It mighttherefore be possible to isolate cells equivalent to certainintermediary differentiation stages from cultures grown for28 days in the absence of glucose. A certain number of suchcells have been isolated; these resemble the original PCC3/A/i embryonal carcinoma cells in their capacity to formtumors containing derivatives of all 3 germ layers.

They differ from these cells, however, in that most of thecell lines: (a) are aneuploid, (b) have a morphology distinctly different from that of EC cells, (c) have lost cornpletely or partially their capacity to differentiate in vitro, (d)have lost alkaline phosphatase and do not have acid phosphatase activity, and (e) some no longer express the F9antigen (Table 1).

The exact relationship of such cell types to those occurring in the normal in vitro differentiation remains to beestablished . However, it is interesting to note that the F9antigen is not obligatorily present on all totipotent cellsderived from teratocarcinomas and that such cells can bemarkedly aneuploid and still retain their capacity to differentiate (5).

Nontumorigenic Variants. It would obviously be of interest to obtain variant EC cells blocked for a specific differentiation. With this aim in view, the EC cells, PCC4, weretreated with nitrosoguanidine (3 @gin Earle's balanced saltsolution), and a large number of clones were isolated andtested for their in vivo differentiation properties (9). A number of nullipotent clones were found. None, however, has asyet been found in which a specific differentiation is blocked.

Some of the clones thus obtained exhibited a greatlyreduced tumonigenicity. Injected under standard conditions(5 x 10@cells) in syngeneic mice, they do not producetumors (9). However, when injected in mice previously Xirradiated (600 A), they form well-differentiated tumors. Inthese cells, some change must have occurred, resulting innew properties, so that now either the cells themselvesstimulate the host rejection mechanisms or are renderedmore sensitive to the host's defense mechanisms.

Conclusion

From this summary, it is clear that different cell types,corresponding to different differentiation states, can beisolated from teratocarcinomas and established in culture.EC cells present a special interest since ultimately they cangive rise, in vitro and in vivo, to all the other cell types. Theyobviously present strong similarities to early embryonicmultipotential cells. Such cells are known to persist in thenormal embryo up to the 5th or 6th day (29, 42). There are

instances, again produce tumors containing derivatives ofthe 3 germ layers.

In some cases, however, the character of the tumor isprogressively modified upon serial transfers. This can resulteither in a modification of the differentiation pattern of thetumor or in a change in the nature of the tumor itself (34,39).

Changes in the Differentiation Pattern of the Tumor. Ouring serial transfer in vivo, progressively different types ofdifferentiation may disappear in some tumors. In most instances, the tumor finally turns into a nondifferentiatedteratocarcinoma (39) from which nullipotential EC cells canbe recovered and established in cultures.

In some rare cases, the tumor keeps some, but not all, ofits original differentiation capacities upon further serialtransfers. For instance, during one of the serial transfers ofPCC3, a tumor was obtained that contained no or very fewmesodermal derivatives. In this particular case, the ascitescontained only cell aggregates and no typical embnyoidbodies. From these tumors, a line of embryonal carcinomatype cells (PCC3/S640) has been established. Like PCC3, itsmodal chromosome number is 40. Reinjected into themouse, these cells always give tumors containing ectodermal and endodermal types of tissues, but never mesodenmalderivatives. Like the original cells, they differentiate in vitrobut, even after 2 months in culture, there is no formation ofcartilage, fat cells, or skeletal muscle.

Changes in the Nature of the Tumor. In some cases, serialtransfer of an originally multipotential ascitic tenatocarcinoma results in the change of the tumor itself. The mostfrequently observed derivation leads towards panietal yolksac carcinoma (PYS) (35). By repeated passaging of embryoid bodies obtained from injection of PCC3, 2 differenttypes of tumors have been obtained . The 1st corresponds toa panietal yolk sac carcinoma. Its cells secrete a basal membnane material, periodic acid-Schiff positive, which comesponds probably to Reichert's membrane. The 2nd is atrophoblastoma. At the beginning of its derivation, onlysmall sectors of tnophoblastic cells were observed in thetumor which contained various types of endodermal cells.As the tumor was transferred, the quantity of the trophoblastic component increased while the periodic acid-Schiffpositive material disappeared. From such tumors, severalcell lines have been established in culture. In vitro, theselines are characterized by the presence of 2 cell types, i.e.,endodermal-like cells and giant cells having variably sizednuclei. Some of the latter type have a morphology stronglyreminiscent of embryonic trophoblast cells. When injectedinto syngeneic mice, these lines form tumors containing the2 types of cells. The ascites which is always present contains many embmyoid bodies of cystic type. It is likely thatthe trophoblastic-type cells in fact derive from the nonsecreting endodermal cells which would, in this case, comespond to cytotnophoblastic cells (Fig. 2d; Table 1).

In these 2 types of tumors, the change in vitro, as in vivo,is irreversible; EC cells never reappear.

ECCellDerivativesObtainedin Vitro

From cultures of EC cells, variants can be obtained in





some reasons to believe that EC cells correspond to suchrather late multipotential embryonic cells, since they do notadhere closely either to blastomeres (C. Babinet and M.Condamine, personal communication) or to inner cell masscells (19) and they carry a surface antigen (PCC4) whichappears only on late inner-cell-mass cells.

Non-EC cell types have so far been isolated at random,either from tumors or from cultures differentiating in vitro. Itis likely, however, that, as more is known about the differentiation process, it will become possible to use selectivemethods to isolate particular cell types.

These cell lines constitute valuable material for differentiation studies. EC cells are especially useful for the analysisof the early embryo; e.g. , they facilitate immunological (3)and biochemical (33) studies which, due to the scarcity ofmaterial, would otherwise remain difficult with the embryoitself. Of further interest regarding EC cells is their capacityto differentiate in vitro. This phenomenon is undoubtedlycomplex. Some aspects of this process are, however, amenable to study.

The non-EC derivatives of teratocarcinomas allow thestudy of later steps of the differentiation process. Some ofthe conditions necessary for the differentiation of epidenmalcells have already been analyzed in such a system (37).

In most instances, the cells obtained after the terminaldifferentiation of EC cells are no longer malignant. Theyappear to present the same characteristics as the homologous cells of a normal embryo. It seems therefore that whatis altered in tematomas is not the capacity to pick and perform a particular program of differentiation. What is alteredis the orderly sequence of events that accompany normaldevelopment. It is the programming of the different programs that is lost, possibly because the normal ordering ofcellular interactions allowed by the normal development ofthe fertilized egg cannot occur.

References

1. Abelev, G. I. Alpha-fetoprotein as a Marker of Embryo Specific Differentiation in Normal and Tumor Tissues. Transplant. Rev., 20: 3, 1974.

2. Artzt, K. , Bennett, 0. , and Jacob, F. Primitive Teratocarcinoma CellsExpress a Differentiation Antigen Specified by a Gene at the T-locus inthe Mouse. Proc. NatI. Acad. Sci. U. S., 71: 811-814, 1974.

3. Artzt, K., Dubois, P., Bennett, D., Condamine, H., Babinet, C., andJacob, F. Surface Antigens Common to Mouse Cleavage Embryos andPrimitive Teratocarcinoma Cells in Culture. Proc. NatI. Acad. Sci. U. S.,70: 2988-2992, 1973.

4. Artzt, K. , and Jacob, F. Absence of Serologically Detectable H-2 onPrimitive Teratocarcinoma Cells in Culture. Transplantation, 17: 633-634, 1974.

5, Avner, P., Dubois, P., Nicolas, J. F., Jakob, H., Gaillard, J., and Jacob, F.Mouse Teratocarcinoma: Carbon Source Utilisation Patterns for Growthand in Wtro Differentiation. Exptl. Cell Res., in press.

6. Baker, R. M., Brunette, D. M., Mankowitz, R., Thompson, L. H., Whitmore, G. F., Siminovitch, L., and Till, J. E. Ouabain Resistant Mutants ofMouse and Hamster Cells in Culture. Cell, 1: 9-21, 1974.

7. Bernstine, E. G., Hooper, M. L., Grandchamp, S., and Ephrussi, B.Alkaline Phosphatase Activity in Mouse Teratoma. Proc. NatI. Acad. Sci.U. S., 70: 3899-3903, 1973.

8. Boon,T., Buckingham,M. E., Dexter,D. L.,Jakob,H.,andJacob,F.TÃ©ratocarcinomede Ia Souris: Isolement et ProprlÃ©tÃ©sde Deux LignÃ©esde Myoblastes. Ann. Microbiol. Inst. Pasteur, 1258: 13-28, 1974.

9. Boon, T., Kellerman, 0., Mathy, E., and Gaillard, J. Mutagenized Clonesof a Pluripotent Teratoma Cell Line: Variants with Decreased Differentiation or Tumor Formation Ability. In: Teratomas and Differentiation, pp.161-169. New York: Academic Press, Inc., 1975.

10. Brinster, R. L. The Effect of Cells Transferred into the Mouse Blastocyston Subsequent Development. J. Exptl. Med., 140: 1049-1056, 1974.

11. Buckingham, M. E., Caput, D., Cohen, A., Whalen, R. G., and Gros, F.The Synthesis and Stability of Cytoplasmic Messenger RNA during Myoblast Differentiation in Culture. Proc. Natl. Aced. Sci. U. S., 1: 1466-1470, 1974.

12. Damjanov, I., and Solter, D. Experimental Teratoma. Current TopicsPathol., 59: 69-130, 1974.

13. Dubbs, D. R., and Kit, S. Effect of Halogenated Pyrimidines and Thymidine on Growth of L Cells and a Subline Lacking Thymidine Kinase.Exptl. Cell Res., 33: 19-28, 1964.

14. Edidin, M., and Gooding, L. R. Teratoma-defined and TransplantationAntigens in Early Mouse Embryos. In: Teratomas and Differentiation, pp.109-121. New York: Academic Press, Inc., 1975.

15. Engelhardt, N. V., Poltoranina, V. S., and Yazova, A. K. Localization ofAlpha-fetoprotein in Transplantable Murine Teratocarcinomas. Intern. J.Cancer, 11: 448-459, 1973.

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17. Fellous, M., Gachelin, G., Buc-Caron, M. H., Dubois, P., and Jacob, F.Similar Location of an Early Embryonic Antigen on Mouse and HumanSpermatozoa. Develop. Biol., 41: 331-337, 1974.

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1976;36:4224-4231. Cancer Res J. F. Nicolas, P. Avner, J. Gaillard, et al. Cell Lines Derived from Teratocarcinomas

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Cell Lines Derived from Teratocarcinomas1tal teratocancinomas, or, like line LT,, from ovarian terato carcinomas (H. Jakob, unpublished results). In contrast, multipotential EC cells