Embryonal carcinoma (EC) cells are the stem cells of teratocarcinomas, and the malignant counterparts of embryonic stem (ES) cells derived from the inner cell mass of blastocyst-stage embryos, whether human or mouse. On prolonged culture in vitro, human ES cells acquire karyotypic changes that are also seen in human EC cells. They also ‘adapt’, proliferating faster and becoming easier to maintain with time in culture. Furthermore, when cells from such an ‘adapted’ culture were inoculated into a SCID (severe combined immunodeficient) mouse, we obtained a teratocarcinoma containing histologically recognizable stem cells, which grew out when the tumour was explanted into culture and exhibited properties of the starting ES cells. In these features, the ‘adapted’ ES cells resembled malignant EC cells. The results suggest that ES cells may develop in culture in ways that mimic changes occurring in EC cells during tumour progression.
- embryonal carcinoma cell
- embryonic stem cell
- germ cell tumour
The concept that cancer arises because of dysfunctional control of stem cells that reside within many adult tissues is an old one. Following the work of Till and McCulloch  who identified the existence of stem cell populations capable of regenerating all cell lineages of the blood, stem cell populations have been identified and studied extensively in many other tissues subject to continual renewal throughout life [2,3]. At the same time, the realization by pathologists that tumours often contain the same range of differentiated cell types as the tissue from which they arise, albeit in a disorganized fashion, promoted the idea that aberrant control of differentiation, as much as aberrant control of cell proliferation, lie at the core of oncogenesis . Cairns  went on to suggest that stem cells are the targets of carcinogenesis and that a tissue architecture based on a small stem cell population compared with a large population of differentiated cells provides one level of protection against the development of cancer. One consequence of these ideas is that, within tumours, the malignant stem cells are rare and perhaps quiescent, so that they may escape the chemotherapy regimens that destroy most of the cells of a tumour, leaving intact those few cells capable of re-initiating cancer development at a later time.
EC (embryonal carcinoma) cells
Teratocarcinomas, a subset of GCTs (germ cell tumours), provide a striking paradigm of the stem cell concept of cancer. They are highly malignant tumours containing a disorganized array of many somatic and extraembryonic cells, together with nests of EC cells. EC cells are the ‘pluripotent’ stem cells of these cancers, capable of self-renewal as well as differentiation into a very wide range of cell types. The differentiated derivatives of the EC cells are typically non-malignant, so that malignancy, as shown by the ability to regenerate the whole cancer including its differentiated elements, is the property of the EC stem cells. This was demonstrated by the classic experiments of Kleinsmith and Pierce  in which they showed that transplantation of a single EC cell to a new host mouse is sufficient to regenerate a new tumour. However, extensive studies during the 1970s also showed a close relationship between EC cells from murine teratocarcinomas and the pluripotent ICM (inner cell mass) cells of the blastocyst stage of early mouse embryos . This, together with an understanding of how to culture and characterize EC cells in vitro, culminated in the isolation of ES (embryonic stem) cell lines by explanting ICM cells from mouse embryos in 1981 [8,9].
The pathology of human and mouse GCTs is significantly different, while the properties of human and mouse EC cells also differ from one another. For example, while trophoblastic differentiation from human EC cells is common, it does not normally occur from mouse EC cells . Furthermore, they express different patterns of characteristic surface antigens. For example, human EC cells typically express the glycolipid antigens SSEA3 (stage-specific embryonic antigen-3) and SSEA4, but not SSEA1, the high molecular mass proteoglycan antigens TRA-1-60, TRA-1-81 and GCTM2 and the protein antigens Thy1 and MHC class 1; in contrast, murine EC and ES cells express SSEA1 but not the other markers . It is noteworthy, then, that when human ES cells were finally derived by explanting the ICM of human blastocysts, their properties closely paralleled those of human EC cells and were distinct from those of mouse ES cells [12,13]. Human ICM cells from blastocysts also express similar patterns of surface antigen expression to both human EC and ES cells, again confirming the relationships between these cell types and emphasizing that the differences from the corresponding mouse cells most probably represent species differences in embryogenesis .
Nevertheless, like murine EC and ES cells, human EC and ES cells characteristically express the transcription factor OCT4, which is down-regulated upon differentiation. Moreover, as in human and mouse ES cells [15,16], knockdown of OCT4 expression in human EC cells using RNAi (RNA interference) techniques results in their differentiation towards trophectoderm (Figure 1). Since OCT4 knockdown also results in a substantial reduction, if not elimination, of cell growth, this observation also suggests a potential therapeutic target for RNAi if an appropriate delivery technology could be developed.
Adaptation and tumour progression
Despite their similarities, teratocarcinoma-derived EC cells present only a caricature of ES cells. In contrast with ES cells, EC cells often only have a limited capacity for differentiation, and many EC cell lines have completely lost this ability – such cell lines are called ‘nullipotent’. Many, particularly in humans, are also karyotypically abnormal . Such differences from ES cells are not particularly surprising when it is appreciated that EC cells have necessarily been selected for tumour growth. While many facets of cell biology might contribute to better survival of tumour cells, a particular feature that is pertinent in the case of pluripotent stem cells is their capacity to choose between the production of daughter stem cells (self-renewal) on the one hand, and differentiation on the other. Since the differentiated derivatives of EC cells have limited competence for extended proliferation and survival, one might infer that pluripotent stem cells like EC cells will be subject to strong selection for mutations that tend to limit differentiation – even a small increase in the probability of self-renewal compared with differentiation could have a significant selective advantage during tumour progression.
If indeed the selection of variants with reduced capacity for differentiation is to be expected in the growth of EC cells, similar selection might also be anticipated in ES cells on prolonged passage in culture. Recently, we have shown that human ES cells in culture commonly acquire additional copies of chromosome 17, particularly its long arm (17q) and also chromosome 12, particularly its short arm (12p) . What is striking about this observation is that the same chromosomal additions are very common in EC cells from human teratocarcinomas [19–21]. Furthermore, amplification of mouse chromosome 11, which is syntenic with much of human chromosome 17q, has also been reported in mouse ES cells , although mouse chromosome 8 is also commonly amplified too. One inference from these observations is that additional copies of a gene(s) encoded by these chromosomes contribute to enhancing the capacity of both EC and ES cells for self-renewal and that such genes encode key components of the molecular mechanism by which these stem cells choose whether or not to commit to differentiation. To date, the identity of these genes remains unknown, although some studies in EC cells have focused on GRB7 as a potential candidate gene that is frequently highly expressed in human EC cells and is located on chromosome 17q .
To explore the issue of culture adaptation of human ES cells further, we have particularly studied a ‘culture adapted’ subline of H7 human ES cells. This cell line originally had a normal human 46,XX karyotype. However, the adapted subline (H7-s6), which shows substantially enhanced growth characteristics, had acquired a translocation of the long arm of chromosome 17 to chromosome 6 [46,XX, der(6)t(6;17)(q27;q1)]; effectively these cells were trisomic for chromosome 17q . Subsequently, on further passage, we have found that the cells acquired an extra copy of chromosome 1, so that their karyotype became 47,XX,+1,der(6)t(6;17)(q27;q1). Generally, human ES cells are cultured in the presence of inactivated feeder cells or in the presence of conditioned medium from such cells. However, from H7-s6, we developed further sublines that retained a pluripotent phenotype even when cultured for a long period without feeders, or conditioned medium, albeit on a Matrigel substrate . One of these sublines was also adapted to grow without feeders and in the absence of FGF (fibroblast growth factor), a growth factor generally thought to be required by human ES cells. Nevertheless, these cells retained pluripotency, expressing typical markers of human ES cells  and produced teratomas when grown in immunosuppressed mice (Figure 2).
One tumour derived from H7-s6 cells also contained cells with the morphological appearance of undifferentiated stem cells – indeed, they resembled EC cells in teratocarcinomas (Figure 2). Furthermore, when this tumour was explanted in culture, cells grew out with the general features of human ES cells, such as the morphology and expression of marker antigens such as SSEA3 and TRA-1-60 (Figure 2). Thus progressive adaptation of human ES cells in culture can result in cells with all the hallmarks of EC cells from teratocarcinomas, while still retaining the capacity for differentiation. The cells in the explanted culture had the same karyotypic changes as the cells injected to form the tumour except that approx. 75% had also acquired an additional copy of chromosome 8 [48,XX,+1,der(6)t(6;17)(q27;q1),+8].
Adaptation of human ES cells after prolonged culture is clearly not surprising. The moment an ICM cell is explanted in vitro it is exposed to a new environment. Its capacity for indefinite proliferation, contrasting with the transitory nature of ICM cells in vivo, is certainly one indication of change. Evidently, any initial change that adapts the cells to in vitro culture is readily reversible, at least in the mouse, since mouse ES cells can contribute to all cell types of a developing embryo, including its germline, when introduced into a blastocyst. But even among mouse ES cells, this capacity may be lost with time, associated with progressive aneuploidy .
In the light of these observations, EC cells from teratocarcinomas and ES cells from embryos might be regarded as existing at different points along a continuous spectrum of adaptation, from complete ‘normality’ at one end (represented by the ICM cell within an embryo) to extreme ‘abnormality’ at the other (represented by a nullipotent EC cell from a GCT). Thought of in this way, ES and EC cells may provide information that is pertinent one to the other, and ES and EC cell lines can provide complementary tools for exploring problems of pluripotency, stem cell biology and cancer. However, the ability of ES cells to undergo progressive adaptation sounds one particular note of caution in relation to experiments to identify key factors that drive their self-renewal. It is quite possible that the cells will have different culture requirements at different stages along the adaptation spectrum – from a requirement for feeders at one end to complete feeder independence, and probably minimal extrinsic factor requirements, at the other. Indeed, as we have found, various sublines of a human ES cell can be isolated with different requirements for feeders and FGF while yet retaining many key features of pluripotent ES cells.
The practical significance for the eventual therapeutic applications of ES cells of this relationship between ES and EC cells, and the phenomenon of adaptation in culture, should not be overstated. It is certain that our current culture techniques are suboptimal. Future developments based upon a firm understanding of the biology of human ES cells and the molecular mechanisms that drive self-renewal and commitment to differentiation will undoubtedly lead to culture methods that will minimize the selective advantage of variant cells. Furthermore, a genetic variant that offers a selective advantage to an undifferentiated ES cell in culture might have no significance for the behaviour and function of a differentiated derivative, although this would clearly need to be demonstrated in any particular case.
However, the phenomenon of adaptation may also be put to good use. ‘Adapted’ ES cells that retain pluripotency may be simpler to use in practical applications such as high-throughput drug screening. More importantly, they may also give us insights into the mechanisms that are involved in controlling stem cell self-renewal, and commitment to differentiation. Finally, since the typical karyotypic changes in adapted ES cells mirror those found in their malignant counterparts from teratocarcinomas, the mechanisms that drive adaptation and selection of such variants in vitro may give insights into the mechanisms underlying GCT progression in vivo. In GCT, the cells are typically grossly aneuploid with many karyotypic changes, many of them being case-specific – only a few of the changes are common to all cases of the tumour and so likely to be relevant to the general mechanisms of self-renewal and differentiation. The relatively few karyotypic changes occurring in adapted ES cells might make analysis of the underlying causes simpler.
We are grateful to many of our colleagues for help and advice and especially Duncan Baker and Kath Smith for karyotype analyses. This work was supported in part by grants from the Juvenile Diabetes Research Foundation, the M.R.C., the BBSRC and Yorkshire Cancer Research.
Stem Cells and Development: A Focus Topic at BioScience2005, held at SECC Glasgow, U.K., 17–21 July 2005. Edited by T. Kouzarides (Cambridge, U.K.), S. Newbury (Newcastle upon Tyne, U.K.), B. Richardson (University College London, U.K.), R. Sablowski (John Innes Centre, Norwich, U.K.), D. Tosh (Bath, U.K.), M. Welham (Bath, U.K.) and A. Willis (Nottingham, U.K.).
Abbreviations: EC, embryonal carcinoma; ES, embryonic stem; FGF, fibroblast growth factor; GCT, germ cell tumour; ICM, inner cell mass; RNAi, RNA interference
- © 2005 The Biochemical Society