/ . Embryol. exp. Morph. Vol. 38, pp. 63-75, 1977 6 3

Printed in Great Britain

Mouse teratomas and embryoid bodies: theirinduction and differentiation

By S. A. ILES1

From the Zoology Department, Oxford

SUMMARY

Teratomas were induced by the transfer of mouse blastocysts (C3H and 129/J strains) andegg-cylinders (C3H) to extra-uterine sites. C3H and 129/J blastocysts cultured in vitro fox: Aor 5 days could also form teratomas in extra-uterine sites.

Four transplantable teratomas, or teratocarcinomas, were derived from C3H embryos;embryoid bodies were derived from each line. The differentiative capacity of a teratocarci-noma was shown to be similar whether it was maintained as a solid tumour or as embryoidbodies.

INTRODUCTION

Both ovarian and testicular teratomas occur spontaneously in certain mousestrains (LT and 129/J respectively); they can also be induced by grafting toextra-uterine sites embryos up to the 8th day of development (129/J, C3H,C57BL, CBA, AKR and A/He strains), or male embryonic genital ridges (129/Jand A/He strains) (reviewed by Solter, Damjanov & Koprowski, 1975). Aproportion of spontaneous (129/J) and embryo-derived (129/J, C3H, A/He and129/J x A/He Fx hybrid) teratomas have been reported to be transplantable fora number of generations (Stevens, 1958, 1970; Damjanov, Solter, Belicza &Skreb, 1971 b); these transplantable teratomas, or teratocarcinomas, containembryonal carcinoma cells (ECC) which are thought to be the stem cells forgrowth at each transplant generation. When 129/J teratocarcinomas are con-verted to the ascites form, the peritoneal fluid contains multi-cellular bodies:these bodies typically consist of a core of ECC, surrounded by endoderm, andare called embryoid bodies (Stevens, 1959; Pierce & Dixon, 1959).

Teratocarcinomas arise only from grafted embryos not older than 8th dayegg-cylinders: older embryos will only form small benign teratomas (Damjanov,Solter & Skreb, 1971 a). Attempts were therefore made to induce tumours fromblastocysts cultured for 4-6 days, so as to investigate (i) if it is the age or thestate of organization of the embryo which determines its ability to form abenign teratoma or a teratocarcinoma, (ii) if a higher incidence of tumours canbe obtained from cultured blastocysts as opposed to untreated blastocysts oregg-cylinders.

1 Author's address: Department of Zoology, South Parks Road, Oxford, 0X1 3PS, U.K.5-2

64 S. A. ILES

Until now, embryoid bodies have only been reported in 129/J mice: this paperdescribes the derivation of embryoid bodies from C3H teratocarcinomas. Acomparison has been made between the developmental capacity of teratocarci-nomas maintained as solid tumours or as embryoid bodies. Such a comparisonprovides information about the relationship between growth conditions and themaintenance of pluripotentiality.

Cell lines have been derived from some of the C3H teratocarcinomas andembryoid bodies described in this paper. Cells from some of the tumours arecapable of colonizing the mouse blastocysts and forming part of the animalwhich is born (Papaioannou, McBurney, Gardner & Evans, 1975).

MATERIALS AND METHODS

(i) Induction of tumours

Blastocysts were flushed from the uterus and teased from the oviducts of C3Hand 129/J females on the 4th day of pregnancy (day of finding the copulationplug= 1st day of pregnancy). Egg-cylinder stages (7th and 8th day of pregnancy)were dissected from uterine decidual swellings and separated from the ecto-placental cone and primary trophoblast. Embryos were transferred beneath thekidney or testis capsule of syngeneic adult recipients with a glass micropipette,controlled by a mouth-piece via a length of flexible polythene tubing. Recipientswere anaesthetized with Avertin (Winthrop, U.S.A.) at 0-01 ml of 2-5 % Avertinper gram body weight. Transfer sites were inspected 2-3 months after transfer,unless stated otherwise.

(ii) Transplantation of tumours and induction of embryoid bodies

Solid tumours were chopped finely with scissors in sterile PBS (Dulbecco 'A 'from Dulbecco & Vogt, 1954). 0-5 ml of this tumour suspension was injectedsubcutaneously or intraperitoneally with a trochar to syngeneic or semi-syngeneic (129/J xC3H Fx) adult recipients under ether anaesthesia. Afterintraperitoneal (IP) passage of a tumour for a few generations, ascites fluid wassometimes found in the peritoneal cavity in addition to solid implants of thetumour. This ascites fluid was found to contain embryoid bodies. The embryoidbodies could be maintained by IP injection of ascites fluid to syngeneic or semi-syngeneic recipients. Solid tumours could also be obtained by subcutaneous(SC) injection of ascites fluid or embryoid bodies washed free of blood andsuspended in sterile PBS.

In the case of short-term growths of 129/J blastocysts in the kidney, the wholegrowth was dissected out of the kidney and transferred to the kidney of anotherrecipient with a wide micropipette.

Induction and differentiation of mouse teratomas 65

(iii) Culture of embryos for transfer

(a) NCTC medium. Blastocysts were cultured overnight in V medium (Whitten,1971) then transferred to drops of NCTC-109 medium (Evans, Bryant, Kerr& Schilling, 1964; Biocult Laboratories, Paisley, Scotland) supplemented with10 % foetal calf serum (FCS) (Chew & Sherman, 1975) under paraffin oil (Boots'liquid paraffin, B.P., U.K.) in glass dishes.

(b) oc medium. Blastocysts were transferred directly to a medium (Stanners,Eliceiri & Green, 1971) supplemented with 10% FCS either in drops underparaffin oil in glass dishes, or in plastic dishes (Falcon Plastics or Sterilin) with-out oil. Some blastocysts were denuded of the zona pellucida with 0-5 % pronaseand incubated in 0-05 % trypsin in PBS for 30 min before culture (Pienkowski,Solter & Koprowski, 1974).

In both (a) and (b), embryos were cultured for a total of 4-6 days in a humidi-fied atmosphere of 5 % CO2 in air at 37 °C. Before transfer to extra-uterine sites,they were detached from the culture vessel with a glass micropipette.

(iv) Histology

Growths were fixed in Bouin's fluid or formol saline, embedded in paraffinwax (M.P. 56 °C) and sectioned at 8 ^m. Sections were stained in alcian blueat pH 2-5 followed by Masson's trichrome (Evans, 1972). One in ten sections ofeach tumour was scanned, but every section of the small growths was inspected.

RESULTS(i) Induction of tumours

(a) 1291J. Ten per cent of 129/J blastocysts (3/28) gave rise to teratomas of atleast half the size of the host organ in the testes, but 129/J blastocysts failed todevelop into tumours of a similar size or tissue composition in the kidney.Seven out of 34 blastocysts transferred to the kidney formed small nodules 1-2mm in diameter. Five of these were fixed and sectioned: one contained an epi-thelial cyst lined by secretory epithelium, while the others contained yolk-sacmaterial, cells resembling trophoblast giant cells and other unidentifiable cells.

In order to investigate this failure of 129/J blastocysts to form teratomas in thekidney, hosts were killed 6 days after blastocyst transfer, and the kidneys wereexamined. In 16 out of 26 recipients, a haemorrhagic spot about 2 mm indiameter was found at the site of transfer. Two of these growths were fixedand sectioned: one consisted of well organized embryonic and extra-embryonicstructures, while the other consisted of trophoblast giant cells and extra-embry-onic tissues. The failure of 129/J blastocysts to form teratomas in the kidneycannot therefore be due to their inability to start to grow in this site.

Ten of these early kidney growths were retransferred to the kidneys of a secondseries of hosts, on the assumption that the disturbance caused by the second

66 S. A. ILES

Table 1. Development of cultured 129\J and C3H blastocysts aftertransfer to the testis

Strain

129/J129/J129/J129/J129/J129/J129/JC3HC3HC3HC3HC3HC3HC3H

Medium

NCTCaaaaaa

NCTCaaaaaa

Pronase andtrypsin

———YesYesYes————YesYesYes

Number ofdays inculture

4-6456456

4-6456456

Number ofblastocysts

2710161313149

521088

18118

Numberof

teratomas

0330300

1310300

(%)

(0)(30)(19)(0)(23)(0)(0)

(2)(30)(12)(0)(17)(0)(0)

transfer might induce the disorganization of ordered embryonic structures thatprecedes teratoma formation (Stevens, 1970). No tumours were found 2 monthsafter second transfer.

It was found that teratomas can sometimes develop from the outgrowthsarising from cultured blastocysts. Blastocysts cultured in NCTC and oc mediahatched from the zona pellucida and attached to the bottom of the culture dish:trophoblast giant cells spread out as a monolayer, while the inner cell mass(ICM) developed into a knob-like structure. This ICM 'knob' often developedinto a two-layered structure resembling an egg-cylinder (as found by Pienkowskiet ah (1974) and Spindle & Pedersen (1973). Outgrowth of blastocysts in amedium was superior to that in NCTC medium. Only embryos with good ICMdevelopment were selected for transfer to the testis, i.e. good proliferation of theICM, but not always with development of two distinct layers.

A proportion of the blastocysts cultured in a medium gave rise to teratomasafter transfer to the testis, but none of the blastocysts cultured in NCTC mediumdid so (Table 1).

(b) C3H. Thirty-six per cent of C3H blastocysts (9/25) formed teratomas inthe testes of adult hosts. Egg-cylinder stages of C3H embryos also gave rise toteratomas in the testis: 33 % of the 7th day embryos (9/26) and 66 % of 8th dayembryos (25/38) formed teratomas. One out of three 7th day embryos transferredto the kidney formed a teratoma.

Outgrowth of C3H blastocysts in culture was similar to that of 129/J blasto-cysts, with superior outgrowth in a medium, but outgrowths were not as largeas those obtained from 129/J blastocysts. A number of blastocysts cultured in

Induction and differentiation of mouse teratomas 67

Table 2. Histology of primary tumours

Origin of tumour

No. of tumours ...ECCNervous tissuePigmentKeratin, epitheliumGlandular epitheliumCiliated epitheliumSimple epitheliumCartilageBoneSmooth muscleStriated muscleAdipose tissueYolk-saccarcinoma

129/J be*

3

lOOf6633

10066

10010033

100100100660

129/Jcult, be

9

33$10055

100100100893333557844

0

C3Hbc

8

75753787877575251262753712

C3H 7thday

embryo

10

609090909080

1008080809040

0

C3H 8thday

embryo

24

6296758775717554547979330

C3Hcult, be

8

75$10062877587876262628725

0

* bc= blastocyst.t Figures in the columns refer to the percentage of tumours showing a particular tissue.% ECC were present mainly in small numbers, and their identification was often uncertain.

a medium gave rise to teratomas after transfer to the testis, whereas only one ofthe blastocysts cultured in NCTC medium did so (Table 1).

(ii) Histology of primary tumours

Table 2 shows the distribution of tissues in the primary teratomas referredto in Section (i) of the Results. Since only part of each tumour was available forhistology (1/20 to 1/100 of each tumour) the absence of a particular tissue fromthe sections examined does not mean that it could not have been present insome other part of the tumour. In a well differentiated tumour, however, mosttissues could be found in the first few sections examined, so the absence of atissue from the available sections makes it very likely that it is absent from therest of the tumour, or only present in very small amounts.

A survey of histology of these primary tumours shows that tumours derivedfrom 129/J blastocysts were not grossly different from those derived from C3Hblastocysts. Some differences could be seen in tumours derived from C3Hblastocysts and those derived from C3H egg-cylinder stages: although a fullrange of differentiated tissues could be found in both types of tumour, moreegg-cylinder-derived tumours showed the full range of tissues than did blasto-cyst-derived tumours (this was particularly noticeable for pigment, cartilageand bone), while yolk-sac carcinoma was found only in a tumour arising froma blastocyst (but see Section (iv) for appearance of yolk-sac carcinoma intransplants of an egg-cylinder-derived tumour).

0 8 S. A. ILES

Table 3. Tissues produced by transplant generations

Generation ...

No. of tumours ...ECCNervous tissuePigmentKeratin, epitheliumGlandular epitheliumCiliated epitheliumSimple epitheliumCartilageBoneSmooth muscleStriated muscleAdipose tissueYolk-sac carcinoma

P

1

It111111111110

1

2

2211222122100

2

4

4424434341400

3*

4

3304422323200

4

3

3202233101102

5

5

5503223121211

6

5

5303233332324

o/ tumour

7

1

1000001110011

8

3

3102113222123

9

1

1000001100000

10

2

2122112221001

P = primary tumour.* Embryoid bodies first appeared in this generation.f Figures in columns refer to number of tumours forming a particular tissue.

Tumours from cultured blastocysts showed a range of tissues similar to thatfound in tumours from untreated blastocysts or egg-cylinders, but ECC, wherepresent, were found only in very small numbers and were hard to identify withcertainty. Tumours from C3H-cultured blastocysts resembled tumours from C3Hegg-cylinders more strongly than they did tumours from C3H blastocysts interms of the extent to which they were differentiated.

(iii) Transplantability of tumours

None of the three primary tumours derived from 129/J blastocysts wastransplantable, but transplantable tumours were obtained from C3H blasto-cysts and egg-cylinders. Not all tumours that grew at the 1st transplant genera-tion continued to grow in subsequent generations, but permanent lines havebeen established from all tumours that continued to grow after the 1st trans-plant. The numbers of C3H tumours still growing are as follows: one out offive tumours from blastocysts transplanted, two out of five tumours from 7thday embryos and one out of 17 tumours from 8th day embryos.

(iv) Progression of transplantable tumours

The histology of the four C3H tumours maintained as transplantable terato-carcinomas has been examined during a number of transplant generations so asto discover whether these would become restricted in their capacity to differen-tiate. Age of tumours is dated from the time of writing (September 1975).

Tumour 17 is derived from a C3H 7th day embryo transferred to the testis2\ years ago; the primary tumour consisted of a wide range of differentiated

Induction and differentiation of mouse teratomas 69

Table 4. Tissues produced by transplant generations of tumour 86

Generation ...

No. of tumours ...ECCNervous tissuePigmentKeratin, epitheliumGlandular epitheliumCiliated epitheliumSimple epitheliumCartilageBoneSmooth muscleStriated muscleAdipose tissueYolk-sac carcinoma

P

1

It111111111100

1

2

2212222222220

2*

222G0000110100

3

2

2200011000000

4

2

2201110000010

5

4

4300003000010

6

3

3312320000000

P = primary tumour.* Embryoid bodies first appeared in this generation.t Figures in column refer to number of tumours forming a particular tissue.

tissues as well as ECC. At the 10th generation, it was still producing all thetissues found in the primary tumour except striated muscle, with the addition ofyolk-sac carcinoma (see Table 3). Yolk-sac carcinoma first appeared in twosub-lines in the 4th generation: it is known that yolk-sac carcinoma can arisefrom embryonal carcinoma cells as well as from embryonic cells from the egg-cylinder (Damjanov & Solter, 1973). In the 5th and 6th generations, some sub-lines produced only ECC and nervous tissue, but only the sub-lines producingECC and a wide range of differentiated tissues were transplanted further. Allthe later generations of tumour 17 were dominated by ECC.

Tumour 86 grew from a C3H 8th day embryo, and has now been maintainedfor If years. The primary tumour contained a wide range of differentiatedtissues together with ECC. During the 2nd transplant generation, a progressivesimplification of the tissues produced occurred. In subsequent generations,tumours consisted mainly of ECC and nervous tissue, together with small areasof epithelial and adipose tissue (see Table 4).

Tumour 106, derived from a C3H blastocyst transferred to the testis If yearsago, progressed in a manner similar to that of tumour 86. Fewer differentiatedtissues were produced in the 2nd and 3rd generations and by the 4th generationECC and nervous tissue predominated, with small areas of epithelium in sometumours (see Table 5).

Tumour 145 was derived from a C3H 7th day embryo transferred to the kidneyonly 8 months ago. The primary tumour contained ECC and a wide range ofdifferentiated tissues. It remained well differentiated in the 2nd and 3rd genera-tions but two sub-lines appeared in the 4th generation: one consisted only of

7U s

Table 5. Tissues produced by

Generation ...

No. of tumours ...EECNervous tissuePigmentKeratin, epitheliumGlandular epitheliumCiliated epitheliumSimple epitheliumCartilageBoneSmooth muscleStriated muscleAdipose tissueYolk-sac carcinoma

P

1

It101111111110

1

1

1111111011100

. A. ILES

transplant

2*

2

2100200001000

generations

3

2

2200101102100

4

5

5501001000000

of tumour

5

4

4400100000000

106

6

4

4400001000000

7

1

1100101000

n10

P = primary tumour.* Embryoid bodies first appeared at this generation.f Figures in the columns refer to the number of tumours forming a particular tissue.% This unexpected appearance of striated muscle in the 7th generation may have been due

to the inclusion of some body wall muscle with the tumour when it was cut out, althoughthis muscle did not have the usual appearance of included body wall muscle.

ECC and nervous tissue with a little ciliated epithelium, while the otherremained well differentiated.

(v) Derivation of embryoid b odies from C3H teratocarcinomas

Intraperitoneal transfer of all four lines of C3H teratocarcinomas alwaysresulted in the formation of ascites fluid containing structures resembling theembryoid bodies arising from 129/J teratocarcinomas (Stevens, 1959, 1970). Intumours 86 and 106, embryoid bodies appeared after IP transfer of 1st trans-plant generation solid tumour, while in tumours 17 and 145, embryoid bodiesappeared one generation later.

These C3H embryoid bodies could be maintained by intraperitoneal passageof ascites fluid (see Methods section). Ascites fluid with embryoid bodies alsoregularly appeared after IP transfer of later generations of solid tumour. Con-versely, implants of solid tumour were often found after transfer of ascites fluidcontaining embryoid bodies.

Transplantability of C3H embryoid bodies appeared to increase with subse-quent generations. For example, the earliest C3H 17 ascitic fluid was trans-planted to 12 recipients: four developed ascites fluid with embryoid bodies,seven failed to develop ascites fluid and one died. Later generations of embryoidbodies were consistently transplantable.

The structure of the embryoid bodies of lines 17, 86 and 106 was studied soonafter their derivation and after at least one year. The early embryoid bodies of

Induction and differentiation of mouse teratomas 71

A , , MJT B

Fig. 1. Embryoid bodies dervied from C3H teratocarcinomas.(A) Embryoid body derived from tumour 17.(B) 'EndodermaP vesicle derived from tumour 17.(C) Early embryoid body from tumour 106.(D) Embryoid body from tumour 86.(E) Embryoid body from tumour 145. Scale bar = 50/*m.

72 S. A. ILES

tumour 17 consisted of masses of ECC and lacked a distinct outer layer; theywere round or oval in cross-section. One year later, their structure was verysimilar (Fig. 1 A). Solid tumour implants found in the peritoneal cavity after IPtransfer of these embryoid bodies give some indication of the potency of theseembryoid bodies: some of these implants from C3H 17 embryoid bodies con-tained ECC and a range of differentiated tissues, others contained ECC, yolk-saccarcinoma, nervous and epithelial tissues, while some consisted only of ECC.More recently, the embryoid bodies of tumour 17 have undergone a distinctchange in morphology: they are now small hollow vesicles of flattened cellsresembling parietal yolk-sac, either empty or filled with material with thestaining characteristics of Reichert's membrane material (Fig. 1B). Injection ofthese embryoid bodies to the subcutaneous space gives tumours consistingentirely of yolk-sac carcinoma.

The early embryoid bodies derived from tumours 86 and 106 were similar instructure to early embryoid bodies from tumour 17, and their structure has notso far changed with time. However, some early embryoid bodies of tumour 106appeared to have a more distinct outer layer of flattened cells (Fig. 1C), resem-bling endoderm. This outer layer was very distinct in some later embryoid bodiesof tumour 86, with some of the inner cells organized into stuctures resemblingearly ectoderm (Fig. 1D). Solid tumours arising after subcutaneous injection ofboth 86 and 106 embryoid bodies containing only ECC and nervous tissue.

Embryoid bodies arising from tumour 145 consisted of masses of ECC, oftensurrounded by a layer of flattened cells (Fig. 1E). Subcutaneous injection ofthese embryoid bodies gave tumours consisting mainly of ECC, together withneuro-ectodermal tubules and simple cuboidal epithelium; these tumoursresembled the subline of tumour 145 in which differentiative capacity wasrestricted.

DISCUSSION

1. Induction of teratomas

C3H embryos form teratomas more frequently as they are transferred to thetestis at progressively later stages of development up to the 8th day. The propor-tions forming teratomas are: no one-cell eggs (lies et al. 1975), one-third of blast-ocysts and 7th day egg-cylinders, and two-thirds of 8th day egg-cylinders. Severalfactors could account for the poorer growth of embryos earlier than the 8th day.Their low cell number could reduce their chances of growing, and their develop-ment may be impeded by haemorrhages resulting from trophoblast growth;more of the trophoblast precursors in the extra-embryonic ectoderm (Gardner& Papaioannou, 1975) are likely to be removed during the dissection of the ecto-placental cone from 8th day embryos, and these embryos grow more frequently.

It is known that the majority of blastocysts transferred to extra-uterine sitesstart to grow (e.g. Kirby, 1963, and 129/J transfers to the kidney in this study).However, the incidence of teratoma formation from blastocysts shows that only

Induction and differentiation of mouse teratomas 73few of these growths develop into teratomas. The culture of blastocystsincreases cell number and disorganizes tissue relationships as the trophoblastlayer spreads out on the substrate: it was therefore hoped that a higher fre-quency of teratoma formation would be obtained from cultured blastocysts.Such an increase would be of value in the induction of teratomas from partheno-genetic blastocysts (lies et al. 1975) which rarely go on to form egg-cylinders invivo. Culture for 4 days in a medium did not raise the incidence of teratoma for-mation by C3H blastocysts above the original one-third, but it increased theincidence with 129/J blastocysts from 10 % (Stevens, 1970, and this study) to30 %; enzymic pre-treatment lowered the incidence of tumour formation fromboth C3H and 129/J blastocysts cultured for 4 days in a medium, while longerperiods of culture greatly reduced tumour formation in both strains. This effectof time is not understood, because embryos of the same total age can formbenign teratomas (Damjanov et al. 1971a). Tumours derived from blastocystscultured for 4 or 5 days do not contain significant amounts of ECC, but they doshow a wide range of differentiated tissues.

2. Development of teratocarcinomas and embryoid bodies

In this study, teratocarcinomas (transplantable teratomas) were obtained fromC3H blastocysts, 7th and 8th day egg-cylinders; similar tumours have beenobtained from C3H 8th day egg-cylinders by Damjanov et al. (1971 b) and from129/J blastocysts and 7th day embryos by Stevens (1970). Both spontaneousand embryo-derived teratocarcinomas may either retain the ability to differen-tiate into many tissues (pluripotent) or their differentiation may be restricted toone or a few tissues (e.g. Stevens, 1958, 1970; Damjanov et al. 19716). Thehistology of these C3H teratocarcinomas on successive transplant generationsshows that 17 retains pluripotency while 86,106 and 145 have become restricted.

The restriction of developmental potential of some tumours may be due tothe selection of rapidly dividing ECC whose capacity to differentiate has be-come restricted: such restrictions may be associated with abnormal karyotypes(see following paper by lies & Evans, 1977). It is important to discover whetherteratocarcinomas maintained as embryoid bodies are more or less restrictedthan those maintained as solid tumours. This paper is the first published descrip-tion of embryoid bodies derived from C3H teratocarcinomas; their structure iscomparable with the simpler of the embryoid bodies in OTT 6050 ascites fluiddescribed by Stevens (1970).

It has been shown here that the developmental capacity cf a teratocarcinomais generally similar whether it is maintained as embryoid bodies or as a solidtumour. Embryoid bodies of 86,106 and 145 all gave subcutaneous tumoursconsisting of embryonal carcinoma and nervous tissue; these tumours stronglyresembled the final state of the solid tumour transplants. For 86 and 106, thisrestriction in developmental capacity could be related to the observation thatsolid tumours arising in the generation in which the embryoid bodies first

74 S. A. ILES

appeared were, in both cases, starting to show a similar restriction in theircapacity to differentiate (see Results); solid tumours of 145 became restricted (inone subline) one generation later than the embryoid bodies first appeared.

Tumour 17 continued to differentiate well, and some solid implants found onthe wall of the peritoneum after injection of embryoid bodies developed a widerange of differentiated tissues. However, the embryoid bodies of 17 have recentlybecome converted from the type of embryoid bodies found in 129/J mice(Stevens, 1959,1970) and formed in vitro from teratocarcinoma cell lines (Martin& Evans, 1975) to sacs of endoderm-like cells, which give rise to yolk-sac carci-nomas when injected subcutaneously. A similar progression to yolk-sac carci-noma has been reported with strain 129/J embryoid bodies (Stevens, 1959;Pierce & Dixon, 1959).

The conclusion from these observations is that tumours may become restrictedin developmental capacity whether they are maintained as solid tumours or asembryoid bodies. Both growth conditions seem to select for cells with rapidgrowth which may have restricted developmental capacities.

I should thank the following people: Dr C. F. Graham, for his advice and constant en-couragement, Dr D. Solter, for reading the manuscript, and S. R. Bramwell, for experttechnical assistance. This work was supported by the Cancer Research Campaign and partlyby a Mary Goodger Scholarship.

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(Received 22 April 1976, revised 21 October 1976)