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Stem Cell Reviews Copyright © 2005 Humana Press Inc. All rights of any nature whatsoever are reserved. ISSN 1550-8943/05/1:273–276/$30.00

Original Article The Road From Teratocarcinoma to Human Embryonic Stem Cells Ivan Damjanov Department of Pathology,The University of Kansas School of Medicine, Kansas

Abstract In this article, a brief review of the research that began with the study of murine teratomas of the testis and ultimately led to the culture of human embryonic stem cells is discussed. Most of the space will be devoted to the studies in which the author personally took part, and the discussion will also touch upon some of the crucial experiments important for the understanding of this entire research effort. Index Entries: Teratocarcinoma; embryonal carcinoma; embryonic stem cells.

Spontaneous Teratomas and Teratocarcinomas

Correspondence and reprint requests to: Ivan Damjanov Department of Pathology, The University of Kansas School of Medicine, 3901 Rainbow Blvd., Kansas City, KS 66160-7410. E-mail: [email protected]

Teratomas are benign germ cell tumors, which in humans occur most often in the gonads, but may be occasionally found in extragonadal sites such as the anterior mediastinum or retroperitoneum (1,2). Histologically, such tumors are made up of various somatic tissues arranged in a haphazard manner. These tissues are thought to be derived from all three embryonic germ layers: ectoderm, mesoderm, and endoderm. Ectoderm gives rise to tissues such as epidermis and neural tissue, mesoderm gives rise to connective tissue, muscles, bones, whereas the endoderm gives rise to intestinal or bronchial-like structures. Teratocarcinomas are malignant germ cell tumors made up of the same somatic tissues as teratomas. In contrast to teratomas, teratocarcinomas contain malignant stem cells, known as embryonal carcinoma cells (ECC). ECC are thought to be malignant equivalents of normal pluripotent embryonic cells from preimplanation stage embryos. This hypothesis is experimentally

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proven in humans many years after it has been proposed (3,4) is based on the fact that the ECC are developmentally pluripotent like the normal embryonic cells. They can also differentiate into many somatic cell types, and under some circumstances also into extraembryonic cell types found in the placenta and the yolk sac. Experimental studies of teratomas and teratocarcinomas became possible in 1950s after Stevens and Little (5) described an increased incidence of teratomas of the testis in 129 mouse strain. Subsequent studies performed at the Jackson Laboratory, Bar Harbor by Leroy Stevens (3) proved that these tumors have a genetic basis, that the incidence of tumors can be increased by appropriate inbreeding of mice, and also that the tumors originate in the fetal gonads from primordial germ cells. In addition to teratomas, Stevens also described several spontaneous tertocarcinomas of the testis, which ultimately led to the isolation of murine ECC (6). Stevens and Varnum (7) described in 1974 spontaneous teratomas in inbred strain LT mice. Biologically these tumors were similar

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Fig. 1. Ovary of an LT mouse. The ovary contains activated germ cells one of which has developed into a blastocyst. Hematoxylin and eosin (H&E) × 280.

Fig. 2. Murine teratoma. The tumor consists of differentiated somatic tissue,such as glands and neural tissue.Hematoxylin and eosin (H&E) ×160.

to those arising from the testis, suggesting a common pathway of tumorigenesis from either male or female germ cells. The studies of the teratomas in LT mice showed that the germ cells in the ovary can be activated parthenogenetically, whereupon the germ cell gives rise to embryonic cells, similar to those formed from a fertilized ovum. Sequential studies of LT ovaries revealed not only activated oocytes, but also 2–16-cell stage embryos and blastocysts (Fig. 1). Blastocysts apparently could not develop further, but the cells derived from the inner cell mass of these blastocysts gave rise to various tissues, which became parts of teratomas developing at a high rate in the ovaries of postpubertal LT mice.

Experimentally Induced Teratomas and Teratocarcinomas On the basis of the morphologic studies performed on murine gonads that gave rise to teratomas and occasionally to teratocarcinomas, Stevens assumed that he could produce teratomas and teratocarcinomas by transplanting mouse blastocysts to extrauterine sites. Similar experiments were performed by Skreb (8) and his associates at the University of Zagreb Croatia, who were transplanting postimplantation mouse embryos underneath the kidney capsule. Interestingly, even

Fig. 3. Murine teratocarcinoma. The tumor contains many condensed groups of embryonal carcinoma cells that appear darker from other tissues. Well-differentiated glands and foci of cartilage are also visible. Hematoxylin and eosin (H&E) × 90.

Fig. 4. Murine teratocarcinoma. In the solid part of the tumor around the dilated duct there are condensed groups of embryonal carcinoma. Hematoxylin and eosin (H&E) × 220.

though both research groups were able to produce teratomas from embryos grown in extrauterine sites, no teratocarcinomas were produced. Apparently, the blastocysts used by Stevens did not survive in extrauterine sites or were destroyed by the trophoblast inducing tissue destruction and hemorrhage. The rat embryos used by Skreb formed readily teratomas (Fig. 2) but were inherently unable to give rise to teratocarcinomas for reasons that remain unknown till today. In the late 1960s Davor Solter and Damjanov joined the laboratory of Skreb and began working with mice instead of rats. Very soon they discovered that malignant teratocarcinomas could be produced by transplanting early postimpalantation mouse embryos underneath the kidney capsule of C3H mice (9). Such tumor contained not only somatic tissues but also undifferentiated embryonic cells (Fig. 3). These embryonic cells grew uncontrollably in the adult host and ultimately killed the host to which they were transplanted. During all that time they retained their undifferentiated embryonic phenotype (Fig.4). In other words these embryonic cells behaved biologically like embryonic carcinoma cells, even though at no point an obvious “malignant transformation” occurred to change either their

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Fig. 5. Embryoid bodies from a murine teratocarcinoma grown in ascites form in the abdominal cavity. The tumor cells form embryoid bodies consists of an inner cell mass and an outer layer corresponding to embryonic visceral endoderm. Hematoxylin and eosin (H&E) × 320.

Fig. 6. Embryoid bodies of a teratocarcinoma examined by electron microscopy. The cells have irregularly shaped nuclei filled with finely dispersed euchromatin and prominent large nucleoli.Osmium and uranyl acetate × 3200.

genotype or phenotype. They concluded that in some conditions the undifferentiated embryonic cells would remain undifferentiated because the adult host to which they were transplanted cannot control their proliferation. Subsequent studies proved that the stem cells of embryoderived teratocarcinomas are indeed equivalent to developmentally pluripotent embryonic cells forming the socalled ectodermal layer of the mouse embryo (egg cylinder) (2). If injected into the abdominal cavity of adult mice they would grow as free-floating bodies reminiscent of embryos and called embryoid bodies (Fig. 5). As suggested by Pierce (4), normal embryogenesis and carcinogenesis of teratocarcinomas were closely interlinked and neoplastic development was just one of the possible pathways for the embryonic cells to take.

mouse and allowed to develop to term. These experiments showed that the ECC readily became parts of the developing embryo and contributed to the cell lineages of many organs. Furthermore, these experiments showed that the malignancy of the ECC could be regulated and completely abrogated. The normal embryonic cells in the blastocyst apparently could annihilate the malignancy of injected ECC. Refinement of these experiments led to a widespread use of EC cells for the production of chimeric mice (12).

Murine ECC Stem cells of teratocarcinoma were cultured in vitro by Martin and Evans (6). Several cell lines were established and many of them remained developmentnally pluripotent. These cells were called embryonal carcinoma (EC) for several reasons: (a) because they were the stem cells of biologically malignant tumors, which killed the host and also could be serially transplanted to other mice and thus propagated ad infinitum; (b) because they could be grown in culture and like other malignant cells, did not show signs of contact inhibition, and could be established as permanent cell lines; (c) because morphologically they resembled human ECC found in human teratocarcinomas (10). By light and electron microscopy these cells had all the features that a well-trained pathologist would recognize as attributes of malignant cells (Fig. 6). Yet, the malignancy of these cells was not irreversible, and if the ECC were treated with retinoic acid their growth could be arrested and they could differentiate into terminally differentiated nonproliferating cells like basement membrane producing parietal yolk sac cells. A major conceptual breakthrough in our thinking about the malignancy of ECC came about in the early 1970s when Brinster (11) succeeded in injecting these malignant cells into the preimplantation stage mouse embryo. The blastocyst carrying the injected ECC was transferred to the uterus of a foster pregnant

Embryonic Stem Cells The fact that teratocarcinomas can be produced from embryos transplanted to extrauterine sites, followed by the discovery that the ECC isolated from these teratocarcinomas can be injected into the blastocysts, motivated several research groups to attempt to produce EC-like cells in vitro directly from the explanted blastocysts. In 1981 Evans and Kaufman (13) and Martin (14) reported almost identical results and showed that indeed embryonic cells can be grown from explanted mouse embryos. These cells, named embryonal stem cells (ESC) resembled ECC in many respects but most importantly, like ECC they were developmentally pluripotent. In contrast to ECC they were not malignant and did not produce tumors when injected into adult hosts. These cells could also be genetically modified and such genetically modified ESCs are currently used for the production of transgenic and knockout mice (12).

Human EC Cell Lines Human teratocarcinomas, also known as mixed germ cell tumors or nonseminomatous germ cell tumors contain ECC, which form groups and even may form some embryo-like structures (Fig. 7). Once the cell culture conditions were defined for growing mouse EC cell lines it did not take long to apply these discoveries to the culture of human ECC in vitro (15). Several cell lines were established, but only a few retained developmental pluripotency (16). Human ECC resemble mouse ECC, but the cells from two species also differ in many aspects (17). Like the murine cells, human ECC can differentiate on exposure to retinoic acid and


276 ___________________________________________________________________________________________________Damjanov cells (19). The work of Thomson and his associates on human ES has opened new avenues for therapeutic cloning and cell therapy, but at the same time it has reinforced the concept of unity between various species (20). By studying murine teratocarcinomas we have over years gathered all the necessary knowledge essential for the study of human cells. The lesson learned from the study of human embryonic stem cells on the other hand, only validates the principles discovered in the murine models.

References

Fig.7.Human embryonal carcinoma cells in a testicular teratocarcinoma. The cells form a structure resembling a malformed early embryo. Hematoxylin and eosin (H&E) × 280.

1. 2. 3. 4. 5. 6. 7. 8.

also can give rise to various somatic tissues both in vitro as well as in vivo on xenografting into nude mice. Most importantly, like the murine ECC, the human ECC can lose their malignancy under appropriate treatment with retinoic acid and transform into nonproliferating terminally differentiated nerve cells (18).

9. 10. 11. 12. 13. 14. 15.

Human Embryonic Stem Cells

16. 17. 18. 19.

The work on mouse teratocarcinomas, murine ECC, murine embryonic stem cells and human ECC established the basis from which it took only a few steps to establish the first primate embryonic stem cells and finally the human embryonic stem

20.

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