JOURNAL OF PATHOLOGY, VOL. 160: 71-76 (1 990)

EXTRAGONADAL TERATOCARCINOMA DERIVED FROM EMBRYONAL STEM CELLS IN

CHIMAERIC MICE KATE H A R D Y * ~ , PHILIP CARTHEW*$, ALAS H. HANDYSIDE*$ AND MARTIS L. HOOPER~

* M RC Espcriniental Ernbrj*ologv and Terutology Unit, M R C Laboratories, Wondniansterne Road. Carshalton, Surrey SM5 4EF, C.K.: $Department ?j’Pnthologj~, Universitj. of Edinburgh, Teviot Place, Edinburgh EH8 9AG, U.K.

Received 24 Jul!. 1989 Revised 15 Septeniher 1989

SUMMARY Three tumours which arose in two (one male and one hermaphrodite) out of 63 chimaeric mice resulting from

injection of E 14TG2a embryo stem (ES) cells into host blastocysts’ have been investigated. All of the tumours appeared within the first 3 weeks after birth. The tumour in the male chimaera and one of the tumours in the hermaphrodite were in the perigenital region but were extragonadal. The third, smaller tumour in the hermaphrodite was on the caecum. The perigenital tumour in the male chimaera was a teratocarcinoma with a wide variety of differentiated tissues, including non-pigmented retina, as well as nests of undifferentiated embryonal carcinoma (EC) cells with high levels of alkaline phosphatase activity. The perigenital tumour in the hermaphrodite was a teratoma, less differentiated and with no evidence of EC cells. Glucose phosphate isomerase isozyme analysis indicated that both perigenital tumours were predominantly of the injected ES cell rather than the host blastocyst type. The possible origins of these turnours, which are the first reported to have arisen from ES cells in chimaeric mice, are discussed.

KEY WORDS-Embryonal stem cells, chimaera, teratocarcinoma, tumour.

INTRODUCTION

Teratomas and teratocarcinomas are germ cell- derived tumours which, in mice, arise with relatively high frequency in the gonads of certain inbred strains and contain a wide variety of differentiated tissues.’ They can also be induced experimentally by grafting early embryos to extra-uterine sites of syngeneic mice. Teratomas are benign and fully differentiated; teratocarcinomas are malignant, transplantable, and, in addition to differentiated tissues, contain nests of rapidly dividing undiffer- entiated embryonal carcinoma (EC) cells. The majority of EC cell lines, derived from terato- carcinomas, are pluripotent and differentiate both in vitro under appropriate conditions and during

Addressee for correspondence: Dr P. Carthew, MRC Toxi- cology Unit. MRC Laboratories, Woodmansterne Road, Carshalton, Surrey SM5 4EF. U.K.

tPresent address: Institute of Obstetrics and Gynaecology, Hammersmith Hospital. Du Cane Road. London W12 OHS, U.K.

:Present address: MRC Toxicology Unit, MRC Laboratories, Woodmansterne Road, Carshalton, Surrey SM5 4EF, U.K.

embryonic development after injection into host blasto~ysts .~-~ However, many of these lines have abnormal karyotypes; contributions to the tissues of chimaeric mice are often small; and chimaeras generated with some EC lines develop tumours of various types in both fetal and adult tissues.6

In contrast, embryonal stem (ES) cell lines estab- lished from preimplantation embryos in ~ i t r o ’ * ~ generally have a normal karyotype and several have been demonstrated to contribute extensively to both somatic tissues and the germline of chimaeras.’ Also, no tumours have been detected in over 500 liveborn chimaeras generated with several ES lines.’’

Here we report the incidence of three tumours, two of which were visible within 3 weeks of birth, in two (one male and one hermaphrodite with an ovo- testis) out of the 63 chimaeric mice which resulted from injection of ES cells of the HPRT-deficient subline, E14TG2a, into host blastocysts.’ Histo- logical examination of the two large perigenital tumours indicated that the tumour in the male was a teratocarcinoma with nests of undifferentiated EC

0022-341 7/90101007146 $05.00 0 1990 by John Wiley & Sons, Ltd.

72 I<. HARDY ET AL.

cells as well as a wide variety of differentiated tissues, and the tumour in the hermaphrodite was a teratonia with mainly mesodermal derivatives. The presence of undifferentiated EC cells in the tumour from the male was confirmed by histochemical localization of high levels of alkaline phosphatase activity." Also, the derivation of these tumours pre- dominantly from the injected ES cells rather than the host embryo was established by analysis of glucose phosphate isomerase isozymes. Finally. the range of differentiated tissues present in these tumours was compared with a tumour resulting from subcutaneous injection of E 14TG2a cells into an adult syngeneic mouse.

MATERIALS AND METHODS

Blastocjet injectioii uiid anulysis of chiinaerus Five to 20 E14TG2a cells were injected into the

blastocoel cavities of host F2 (CBA!CaLac x C57B116JLac) blastocysts. Injected blastocysts were allowed to re-expand in culture and were transferred to pseudopregnant recipients. Two genetic markers were used to distinguish contributions from the host blastocyst and the injected ES cells in chimaeric mice: F2 blastocysts are wild type at the albino locus ( C C ) and homozygous for the fast migrating iso- z 'me of glucose phosphate isomerase (GPI) (Gpi-

and homozygous for the slow isozyme Gpi- 1 "Gpi- I". Out of 63 chimaeras, identified initially by coat colour 5 days after birth,' one male and one externally female chimaera which developed tumours in the perigenital region at 2-3 weeks of age were autopsied at 5 and 10 weeks. respectively. Tumours were dissected into three parts for cryostat sectioning and alkaline phosphatase histo- chemistry, paraffin sectioning, and histological examination for various differentiated tissues and GPI isozyme analysis.

1 x , iGpi-Ib); ' El4TG2a cells are chinchilla (CchiCch)

Histology Cryostat sections-The tissue was snap-frozen in

liquid nitrogen and sectioned in a cryostat. Sections were stained for alkaline phosphatase activity using the standard histochemical method."

Parafin sections-The tissue was fixed in 10 per cent phosphate-buffered formal saline for vacuum embedding in paraffin wax and 5pm paraffin sec- tions were stained by one of three methods: (i)

haematoxylin and eosin for general tumour mor- phology; (ii) a combination of the periodic acid Schiff technique and Alcian blue to stain mucins; and (iii) Palmgren's silver staining method for nerve fibres. '' Isozyme unulj~sis

The GPI isozymes of freeze/thawed tumour samples were analysed by cellulose acetate electro- phoresis. staining. and laser densitometry.13

Suhcuranrous El4 TG2u tumours The spectrum of differentiated tissues present in

the tumours from the chimaeras was compared with that of a tumour generated subcutaneously by injected ES cells of the same subline into an adult mouse. lo6 El4TG2a cells suspended in 0.5 ml cul- ture medium were injected subcutaneously into the flank of a syngeneic 129/01a female mouse. The resulting tumour was passaged in vivo by inserting a small piece through an incision in the skin over the flank of each of three other mice. The remaining part of the tumour was fixed and prepared for histology as above.

RESULTS

Out of 63 coat colour chimaeras, two (one male and one externally female) developed large tumours in the perigenital region. The tumours were first observed at 2 weeks of age and grew progressively to a diameter of between 3 and 4cm when the mice were killed (the male at 5 weeks and the other animal at 10 weeks). Although both tumours were close to the gonads, careful inspection during dissection confirmed that they were extragonadal. Histologi- cal examination of the left gonad of the externally female chimaera. which was located lower down in the abdomen close to the perigenital tumour, revealed that it was an ovotestis with 70-80 per cent testicular tissue (although they were no mature spermatozoa). This externally female chimaera was, therefore, a hermaphrodite. The female also had a small tumour on the caecum which was not analysed further. With both of the tumours in the perigenital region, electrophoretic analysis of isozymes of GPI indicated that they were predominantly (85 per cent) derived from the E14TG2a ES cells. The minor contribution from the host tissue in these tumours could probably be accounted for by connective tissue or blood. However, we cannot exclude the

EMBRYONAL STEM CELL TUMOURS 73

possibility of induction of tumorigenicity in the host embryo.

The perigenital tumour in the male chimaera was a teratocarcinoma with a wide variety of tissues derived from all germ layers as well as groups of morphologically undifferentiated cells with high levels of alkaline phosphatase activity (Fig. I ) . Differentiated tissues included large amounts of neural tissue and several retina-like structures with- out melanocyte layers (Fig. 2). In one case, nerve fibres emanated from the ganglion layer and eathered together to form a nerve bundle. In iddition to fully differentiated nervous tissue. there were also patches of primitive neuroepithelium which resembled the neural tube in the mid-gestation mouse embryo. Other tissues of ectodermal origin included squamous keratinizing epithelium and hair follicles containing hair shafts. Mesodermal deriva- tives were represented by loose connective tissue, muscle, adipose tissue, cartilage, and bone. Cartilage and bone tissues were often continuous (Fig. 3). Epithelia of endodermal origin included ciliated respiratory epithelium and mucin-secreting intesti- nalepithelium with villi. It was very noticeable that sharp transitions in epithelial cell type from keratin- izing squamous to columnar with cilia and columnar

Fig. I-Nests ofalkaline phosphatase-positive, undifferentiated. embryonal carcinoma cells present in the teratocarcinoma

wit; mucin-secreting cells often occurred over a short distance in the epithelia of this tumour (Fig. 4). The perigenital tumour from the hermaphrodite was a cystic teratoma with predominantly meso- dermal tissues. No areas of undifferentiated cells or groups of cells with high alkaline phosphatase activity were detected.

Subcutaneous injection of E14TG2a ES cells into the flank of a syngeneic female resulted in the development of a large tumour after 1-2 months. Although this tumour contained a variety of differ- entiated tissues, there were also large areas of undifferentiated cells with high levels of alkaline phosphatase activity. A detailed comparison of the tissue and cell types present in this subcutaneous tumour and the perigenital tumours in the chimaeras is presented in Table I. Small portions of this tumour were transplanted subcutaneously to the flanks of a further three females, and in one of these a small tumour developed which showed a similar pattern of differentiation to the parent tumour.

DISCUSSION

The three E 14TG2a-derived tumours described here are the first reported to have arisen in chimaeric Fig. 2-Retinal structure formed in the teratocarcinoma

74 K. HARDY E T A L .

Fig. 3-Elements of endochondrial bone formation showing Fig. M o m p o s i t e epithelium with abrupt changes from ciliated cartilage on one side graduating to functional bone with columnar to mucin-secreting to squamous keratinizing haemopoietic activity epithelium

mice resulting from the injection of ES cells into host blastocysts. Previously no tumours were detected in over 500 chimaeras generated from over 17 different ES cell lines.* The teratocarcinoma is unusual in that it contains retinal elements which have been found in transplanted ovarian teratocarcinomas examined previ~usly. '~ but not in any of the extremely rare extragonadal teratomas previously reported." The incidence of tumours is low; only two out of 63 (3 per cent) coat colour chimaeras developed tumours during the first 3 months after birth. But the overall incidence may be higher since any tumours arising during fetal development which caused prenatal mortality or losses at birth would not have been detected. Two more tumours have been detected recently in much older chimaeras at autopsy (personal communication from C. E. Patek). However, these tumours are distinct in that they are not teratocarcinomas, each being com- posed of a single cell type, and it has not yet been established whether they are of ES cell origin.

In contrast to ES cells, tumours frequently arise in chimaeras resulting from the injection of EC cells into blastocysts, both during embryogenesis and in the adult, and contributions to normal tissues are often small or undetectable. However, many of

these EC lines were derived originally from a small number of teratocarcinomas serially passaged in vivo and have grossly abnormal karyotypes with a high frequency of aneuploidly.'6 The few euploid EC lines which have been isolated contribute more frequently and to a larger extent to the normal tissues of chimaeras. Other euploid EC lines, never- theless, give rise to a high incidence of tumours in chimaeras. In these cases, i t is assumed that genetic defects that are not detectable cytologically may be responsible. ''."

The ES line El4 and the HPRT-deficient subline E14TG2a (at a similar passage number to those injected) have normal 40,XY male karyotypes, a majority of cells possessing the modal number of 40 chromosome^.'^ Also, the high frequency and extent of contributions to the normal somatic tissues and germline of the EI4TG2a cells in these chimaeras' were similar to those reported for several other euploid ES lines." This indicates that this subline, although known to have a deletion in the X chromo- some removing the first two exons of the HPRT gene."." is otherwise not carrying any genetic defects affecting totipotency. On the other hand, cells with genetic abnormalities are likely to arise spontaneously at low frequency with any cell line

EMBRYONAL STEM CELL TUMOURS 75

Table I-Comparison of the tissue types in the perigenital turnours of the tho chimaeras with those present in tumours arising from subcutaneous injection of E14TG3a ES cells into 129 Ola mice

Tissue type

Perigenital turnours ES cell from chimaeras tumour

Male Female in v i ~ o

Undifferentiated embryonal carcinoma cells (alkaline phosphatase positive)

Ectodermal derivatives Primitive neuroepithelium Mature neural tissue Keratinized epithelium Hairs and follicles

Mesodermal derivatives Connective tissue Cartilage Bone Bone marrow Muscle Adipose tissue

Endodermal derivatives Ciliated epithelium Gut epithelium Mucus-secreting epithelium

Organoids and others Epithelial cysts Retina Glandular epithelium (acinar)

+

+ + + +

+ + + + + +

+ + +

+ + +

-

-

+ + -

+ + + + + -

+ + +

+ + -

+

+ + + -

+ + + + + +

+ + +

+ + -

maintained in vitro and this could explain a low incidence of tumours. However, there were sub- stantial normal contributions to the coat and other tissues in both tumour-bearing chimaeras, and the range of differentiated tissues in the perigenital teratocarcinomas from the male chimaera was very similar to those present in a subcutaneous tumour resulting from the injection of E14TG2a cells into an adult syngeneic mouse (Table I).

Alternatively, the tumours may result from a saturation of the capacity of the blastocyst to regu- late euploid ES cell development. This possibility is supported by observations on blastocysts injected with various numbers of EC cells and subsequently allowed to outgrow in vitro." When only a few cells from particular EC lines were injected, they were incorporated into the developing egg cylinder; whereas if larger numbers of cells were injected,

some EC cells appeared to 'escape' the regulative influence of the normal ICM cells and continued to proliferate as undifferentiated EC colonies. Since we were often injecting large numbers of E 14TG2a cells (up to 20 cells) into host blastocysts, the regulative capacity of the ICM may have been exceeded. On day 5 between 102 and 114 h post-coitum, there was an average of 2 1-26 ICM cells out of a total of 11& I19 cells in late blastocysts from an outbred mouse strain." But of this minority of ICM cells, only a proportion forms the epiblast or embryonic ecto- derm which gives rise to the tissues of the fetus.24 If one or a few ES cells did not participate in normal embryogenesis but remained viable, these cells could then form tumours in the neonatal chimaeras. Thus, the complete absence of tumours with other ES cell-derived chimaeras may simply be attribut- able to the injection of fewer (8-12) ES cells."

76 K. H A R D Y ET AL.

Injection of blastocysts with various numbers of cloned and karyotypically normal ES cells which are either allowed to outgrow i j z vifra or develop to term iiz vivo after transfer to recipients should allow this to be tested.

A C K N O W L E D G E M E N T S

We would like to thank A. Verstraete and Mrs Jennifer Edwards for assistance with histology, S. Dolan for supervision of the chimaeric mice. and Mrs G. Zdaniecki for typing the manuscript.

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