The Tumorigenicity of Human Embryonic Stem Cells

The Tumorigenicity of Human Embryonic Stem Cells Barak Blum and Nissim Benvenisty Stem Cells Unit, Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem 91904, Israel

I. Introduction II. Spontaneous and Experimental Teratomas and Teratocarcinomas A. Spontaneous Teratomas and Teratocarcinomas B. Experimental Teratomas, Teratocarcinomas and ES Cells C. Definition of Experimental Teratomas and Teratocarcinomas III. Cellular and Molecular Aspects of HESC Tumorigenicity A. In Vivo Differentiation of Embryonic Carcinoma Cells B. Culture Adaptation of HESCs In Vitro C. Molecular Biology of Culture Adaptation in HESCs D. Tumorigenicity of Nonadapted HESCs IV. HESC‐Induced Teratomas as a Model for Early Human Development A. Modeling Normal Embryogenesis B. Modeling Genetic Diseases C. Utilizing HESC‐Induced Teratomas as a Surrogate Human Environment for Cancer Research V. HESC‐Induced Teratomas as a Clinical Hurdle A. General Ablation of Teratoma Cells B. Differentiation to Eliminate Tumorigenic Cells C. Sorting for Nontumorigenic Populations or against Pluripotent Cells VI. Concluding Remarks References Human embryonic stem cells (HESCs) are the in vitro descendants of the pluripotent inner cell mass (ICM) of human blastocyst stage embryos. HESCs can be kept undifferentiated in culture or be differentiated to tissues representing all three germ layers, both in vivo and in vitro. These properties make HESC‐based therapy remarkably appealing for the treatment of various disorders. Upon transplantation in vivo, undifferentiated HESCs rapidly generate the formation of large tumors called teratomas. These are benign masses of haphazardly differentiated tissues. Teratomas also appear spontaneously in humans and in mice. When they also encompass a core of malignant undifferentiated cells, these tumors are defined as teratocarcinomas. These malignant undifferentiated cells are termed embryonic carcinoma (EC), and are the malignant counterparts of embryonic stem cells. Here we review the history of experimental teratomas and teratocarcinomas, from spontaneous teratocarcinomas in mice to induced teratomas by HESC transplantation. We then discuss cellular and molecular aspects of the tumorigenicity of HESCs. We also describe the utilization of HESC‐induced teratomas for the modeling of

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0065-230X/08 $35.00 DOI: 10.1016/S0065-230X(08)00005-5

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early human embryogenesis and for modeling developmental diseases. The problem of HESC‐induced teratomas may also impede or prevent future HESC‐based therapies. We thus conclude with a survey of approaches to evade HESC‐induced tumor formation. # 2008 Elsevier Inc.

I. INTRODUCTION Human embryonic stem cells (HESCs) are pluripotent cells derived from the inner cell mass (ICM) of a human blastocyst stage embryo (Thomson et al., 1998). They are characterized by their ability to self‐renew by cellular divisions and their ability to differentiate to all somatic tissue of the embryo (pluripotency). HESCs grow in tightly packed colonies and, if supplied with specific culture requirements such as a supportive feeder layer (usually mitotically arrested mouse embryonic fibroblasts), can remain undifferentiated indefinitely. HESCs are also defined by the expression of a battery of typical genes, the most renown among them are Oct4, Nanog, Sox2, high telomerase activity, and typical cell surface markers such as SSEA3, SSEA4, TRA‐1–60, TRA‐1–81, and tissue‐specific alkaline phosphatase (Adewumi et al., 2007). HESCs currently open some of the most promising avenues in the field of regenerative medicine, and efforts are being made to establish HESC‐based therapy for various diseases such as Parkinson’s disease, heart failures, and diabetes (Blum and Benvenisty, 2005). Accordingly, reports on the successful differentiation of HESCs to CNS neurons, cardiomyocytes, insulin‐secreting cells, and many other cell types are rapidly accumulating (Blum and Benvenisty, 2005). HESCs can spontaneously differentiate in vitro in the form of embryoid bodies (EBs) (Itskovitz‐Eldor et al., 2000) (Fig. 1). However, HESCs ability to spontaneously differentiate is best manifested when these cells are transplanted in vivo into immunosuppressed mice, where they form typical gross looking tumors termed teratomas, in which the cells differentiate disorderedly to various tissue types of the embryo (Przyborski, 2005) (Fig. 1). This tumorigenic nature of HESCs is considered a major hurdle for their clinical utilization, but it can be valuable for other purposes, such as studying early human development. This tumorigenic nature of HESCs is also important for the assessment of the differentiation potential of newly derived pluripotent cells, since blastocyst injection of HESCs is obviously impractical (Lensch et al., 2007). Almost two decades before HESCs were first successfully derived from human embryos (Thomson et al., 1998), the first mouse embryonic stem (ES) cells were derived (Evans and Kaufman, 1981; Martin, 1981). These successful establishments of blastocyst‐derived ES cells are based on work previously performed on pluripotent cells that were isolated from teratocarcinomas

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In vivo differentiation

1 cm Teratoma

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50 mM Embryoid bodies

Fig. 1 HESCs spontaneously differentiate in vitro in the form of EBs (right), and in vivo in the form of a teratoma (left).

(Andrews, 2002; Damjanov, 2005; Solter, 2006). These tumor cells are very close counterparts of HESCs because of their ability to self‐renew and to differentiate in culture. HESCs are unique in that they are tumorigenic yet perfectly normal in every other aspect. They thus can make an excellent tool for the understanding of tumorigenicity. Accordingly, HESC and many tumor cells hold some similarities, such as the aforementioned self‐renewal and undifferentiated phenotype, along with the expression of telomerase, the ability for in vivo angiogenesis, shortened cell cycle and, of course, the ability to generate tumors upon transplantation in vivo (Dreesen and Brivanlou, 2007). In this review we will discuss the relations between HESCs and their tumor counterparts, outlining the differences and similarities between them. We will also discuss cellular and molecular aspects of HESC tumorigenicity, the use of HESC‐induced tumors for studying human embryonic development, and conclude the discussion by reviewing some of the approaches for evading the appearance of these tumors in clinical utilization.

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II. SPONTANEOUS AND EXPERIMENTAL TERATOMAS AND TERATOCARCINOMAS A. Spontaneous Teratomas and Teratocarcinomas Spontaneously occurring teratomas and teratocarcinomas represent a unique set of tumors. They are categorized among the group of germ cell tumors (GCTs), and are characterized by the presence of haphazardly arranged differentiated tissues representing the three embryonic germ layers. This points to their origin from a pluripotent precursor (Ulbright, 2005). GCTs appear both in gonadal and extragonadal sites along the body midline, and are classified into five pathological groups (Looijenga et al., 2007; Oosterhuis et al., 2007). Thus, type I GCTs are teratomas and yolk sac tumors of infants, which mostly occur on extragonadal sites; type II are seminomas and nonseminomas (amidst which are both teratomas and teratocarcinomas), which occurs mainly in the testes of young adult males, and also occasionally on extragonadal sites along the midline of the body (along the trail of germ cell migration during embryogenesis). Type III GCTs are spermatocytic seminomas, which occur only in the testes of adult males. Type IV are dermoid cysts, and type V are hydatiform moles, both occurring only in females. Hence, teratomas can be classified as type I or type II GCTs. On the basis of their site of appearance, the age and gender of the patient, the repertoire of the cells that comprise them, and the cytogenetic (i.e., karyotype) and epigenetic (i.e., genomic imprinting, gene expression) characteristics of the tumor, the supposed cell of origin of each of these neoplasms can be traced (Looijenga et al., 2007; Oosterhuis et al., 2007). According to this classification, the authors propose a different cell of origin for different tumor categories. For example, while type I teratomas are benign and possess normal diploid karyotype, teratomas and teratocarcinomas categorized within type II nonseminomas are aneuploid (Codesal et al., 1991; Mayer et al., 2003; Looijenga et al., 2007). It is thus hypothesized that teratomas classified as type I GCTs originate from a cell akin to ES cell whereas the teratomas or teratocarcinomas classified as type II GCTs originate from a cell closer to a primordial germ cell (PGC) (Looijenga et al., 2007).

B. Experimental Teratomas, Teratocarcinomas and ES Cells In 1954, Stevens and Little reported that a certain mouse strain, named 129, is specifically prone to develop testicular GCTs (Stevens and Little, 1954) (Fig. 2). Later, they and others were able to produce experimental

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ICM/ES cells

Egg cylinder stage embryo

Genital ridge

1 cm

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Fig. 2 Sources of teratoma formation in mouse and human. Teratomas can be experimentally induced from ES cells, whole embryos at the egg cylinder stage or genital ridges of embryos between E11 ando E13.5. Teratomas also occurred spontaneously in both mouse and human. Note the many differentiated structures in the histological section.

teratocarcinomas by transplanting the undifferentiated core of the tumor back into the mouse, demonstrating the pluripotent tumor initiating cell, termed embryonal carcinoma (EC) (Stevens, 1958; Kleinsmith and Pierce, 1964). EC cells were observed to be localized in small foci (“nests”) of morphologically undifferentiated embryonic like cells. Most EC cells were

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demonstrated to have an abnormal karyotype, usually in the form of near diploid aneuploidity (McBurney, 1976; McBurney and Rogers, 1982). It was also observed that tumors that did not contain these EC nests were never transplantable, grew slower, and eventually ceased to grow (Stevens and Hummel, 1957; Stevens, 1959; Martin, 1975). On the other hand, tumor fractions containing EC cells were always transplantable. When single EC cells were transplanted, the resulting tumor was not composed of EC cells only, but was again a teratocarcinoma containing many differentiated tissues (Kleinsmith and Pierce, 1964), proving the pluripotency of the EC cells. Accordingly, some EC lines grew in culture as two distinct types of colonies. While one type had a fibroblastic phenotype and did not form tumors, the other type grew as tight colonies of undifferentiated cells that could give rise to both types upon clonality assays, and could form complete teratocarcinomas in vivo (Martin and Evans, 1974). Teratomas and teratocarcinomas were also experimentally formed by ectopic transplantations of normal mouse embryos or embryonic genital ridges to adult host, usually beneath the kidney capsule (Solter and Damjanov, 1979; Solter et al., 1970; Stevens, 1964, 1967, 1968; Stevens and Hummel, 1957) (Fig. 2), demonstrating the existence of tumor initiating cells also in normal embryos. These experimental teratomas were dependent upon the age of the transplanted embryos. Thus, teratomas could be obtained only from mouse embryos younger than 7 days (Damjanov et al., 1987) and from genital ridges of embryos between embryonic day 11 and 13.5 (Andrews, 2002). In common to these time points is the fact that after this time, no pluripotent cells are present in the transplant (presumably primitive ectoderm in the early embryos or PGCs in the genital ridges). EC cells were also isolated from spontaneous human GCTs and were characterized (Andrews et al., 1984). Notably, these cells differ in several aspects, mainly cell surface antigens, from mouse EC cells (Andrews, 2002; Andrews et al., 1987). In 1981, pluripotent ES cells were isolated from mouse embryos (Evans and Kaufman, 1981; Martin, 1981). These ES cells appeared similar to most mouse EC cells in their cell surface markers and in their growth requirements. However, differently from EC cells, the new ES cells were karyotypically normal, and displayed broader capacity to differentiate (Andrews, 2002; Bradley et al., 1984). Since 1995, ES cells were established from nonhuman primates (Thomson et al., 1995, 1996), and finally, in 1998, from human embryos (Thomson et al., 1998). These human ES cells were slightly different from mouse ES cells in their growth requirements and the expression of some, but not all, cell surface molecules, but resembled very closely the human EC cells. This was evident by the expression of specific pluripotency markers like SSEA3, SSEA4, TRA‐1–60, TRA‐1–81, telomerase, and alkaline phosphatase, and by the ability to form teratoma in immunodeficient mice (Thomson et al., 1998). In later years, human ES

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and EC cells were also compared for gene expression using microarray analysis (Liu et al., 2006; Sperger et al., 2003), the results showing many similarities, but also noticeable differences. Thus, human ES cells are the nonmalignant equalities of human EC cells, whereas mouse ES cells are the nonmalignant counterparts of mouse EC cells. This difference in some patterns between mouse and human ES and EC cells, along with the notion that GCT pathogenesis probably differs between mouse and human (Clark, 2007; Oosterhuis and Looijenga, 2005; Walt et al., 1993), has led to the suggestion that HESCs may come of an utterly different origin than the mouse ES cells, and are not, after all, truly equivalent interspecies counterparts (Zwaka and Thomson, 2005). With some species, like the rat, for example, it has been consistently very hard to obtain ES cells from (Skreb and Svajger, 1975). Intriguingly, these species had also much less‐reported incidents of spontaneous GCTs (Damjanov, 1993; Damjanov et al., 1987).

C. Definition of Experimental Teratomas and Teratocarcinomas There is much confusion regarding the terminology of teratoma/teratocarcinoma in the experimental setting, partially owing to inconsistencies in the use of medical terminology (Damjanov and Andrews, 2007; Lensch and Ince, 2007). From a histopathological point of view, benign GCTs with differentiation to all embryonic germ layers are termed “teratomas.” These can be mature teratomas (which contain only mature, well‐differentiated tissues) or immature teratomas (which contain tissues of more embryonic, less‐differentiated nature). If the tumors also contain clusters of totally undifferentiated, highly malignant embryonic carcinoma (EC) cells, than they are defined as “teratocarcinomas” (Gonzalez‐Crussi, 1982; Pierce et al., 1960). The presence of EC cells is currently best detected histologically by their immunopositivity for the expression of Oct4 (Jones et al., 2004).

III. CELLULAR AND MOLECULAR ASPECTS OF HESC TUMORIGENICITY A. In Vivo Differentiation of Embryonic Carcinoma Cells As is known from every knockout mouse thus far made, normal euploid mouse ES cells lose their tumorigenicity by incorporation into age‐ complemented embryonic environment. This is conclusively manifested by their incorporation into the blastocyst to form completely normal, germ line

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transmitting chimera (Bradley et al., 1984). Aneuploid mouse teratocarcinoma‐ derived EC cells, however, could not be completely reversed by injection to the blastocyst. It appeared that some of the chimera progeny develop tumors (mostly embryonic carcinoma) and are not germ line competent (Hochedlinger and Jaenisch, 2006; Papaioannou et al., 1975; Rossant and McBurney, 1982). In a straightforward approach, Blelloch et al. (2004) determined the developmental capacity of several mouse teratocarcinoma cell lines to contribute to a normal chimera. They have performed cloning by nuclear transfer of three mouse teratocarcinoma cell lines, and produced ES cells from the resulted blastocysts. Strikingly, the derived ES cells were identical in their differentiation potential to the parental EC cells, demonstrating that the reduced differentiation potential of an aneuploid EC cell cannot be overruled. In this experiment, the only EC line that could still contribute to all embryonic tissues was found to be karyotypicaly normal, and was thus an exception to the rule. Similarly, human EC lines vary in their differentiation capacity between partial pluripotency to complete nullypotecy (Andrews et al., 1987). Hence, aneupolidity in EC cells is deleterious to their ability to differentiate, and the more genomic alternation a cell has, the less differentiation capacity is displayed by this cell. More importantly, and as mentioned earlier, the aneuploidity of EC cells also reflects the malignant nature of teratocarcinomas.

B. Culture Adaptation of HESCs In Vitro When first isolated, HESCs were thought to maintain normal karyotype for many passages in vitro (Amit et al., 2000; Thomson et al., 1998). However, it has been subsequently found that karyotypic changes do occur, the rate of being attributed by many to be dependent on passage number. Gain of chromosomes 12, 17, and X is frequently observed, but other karyotypic changes have also been reported (Baker et al., 2007; Draper et al., 2004; Imreh et al., 2006). In some HESC lines that were grown extensively to high passage, small genomic aberrations that were not apparent on regular G banding karyotyping were discovered using genomic array methods (Maitra et al., 2005). These were also accompanied with aberrations in mitochondrial DNA and impaired imprinting, all previously associated with cancers. Enver et al. (2005) have compared gene expression in a single HESC line before and after culture adaptation. Their results indicate that insufficient X chromosome inactivation is another way by which culture‐adapted HESCs can virtually obtain an additional copy of this chromosome even without apparent genetic changes. Interestingly, the same karyotypic abnormalities that accompany culture‐ adapted HESCs are also frequent in human GCTs and human teratocarcinoma

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cell lines (Almstrup et al., 2006; Andrews et al., 2005; Baker et al., 2007). Particularly observed in these tumors is the gain of an isochromosome 12p (Dal Cin et al., 1989; de Bruin et al., 1994; Speleman et al., 1990), but also the addition of the X and 17 chromosomes has been reported (Kraggerud et al., 2002; Looijenga et al., 1997). An interesting finding, further stressing the similarity between culture‐adapted HESCs and EC cells, is the discovery of Herszfeld et al. (2006) that a specific EC surface antigen, CD30, is exclusively expressed on culture‐adapted, karyotypically abnormal HESCs, but not euploid low passaged cells. Mouse ES cells were also reported to acquire chromosomal changes during high passage adaptation in culture. These changes were directly correlated to their differentiation potential, as cells harboring them were impaired in contributing to germ line transmitting chimera, compared to euploid counterparts (Liu et al., 1997; Longo et al., 1997). Blastocyst injection of normal or culture‐adapted HESCs to asses their differentiation potential could obviously not be performed. However, their capacity to differentiate and their degree of malignancy can be studied through the hisopathological examination of the tumors that they make in immunodeficient mice. As expected from aneuploid mouse EC and ES cells studies, it was shown that culture‐adapted HESCs that were injected into immunodeficient mice develop tumors of a less‐differentiated nature. This was evident by the less mature tissues in them and the detection of undifferentiated nests of cells resembling EC (Andrews et al., 2005) and was most prominent in the works of Herszfeld et al. (2006) and Plaia et al. (2006). They have compared the tumorigenicity of normal unadapted HESCs to the tumorigenicity of CD30‐positive HESCs harboring trisomy in various chromosomes (Herszfeld et al., 2006; Plaia et al., 2006) and found that the karyotypically abnormal cells generated tumors with much primitive, undifferentiated tissues. Plaia et al. (2006) also reports on clusters of Oct4 expressing cells within the tumor. It should be mentioned, however, that there is a single work which specifically reported that HESCs‐bearing trisomy of chromosome 12 did not exhibit more aggressive or less mature teratomas than wild type cells (Gertow et al., 2007), but the differentiation was somewhat biased toward mesodermal lineages.

C. Molecular Biology of Culture Adaptation in HESCs The karyotypic instability of HESCs in culture could be related to an uncoupling between the G2/M checkpoint and the apoptosis machinery. It was recently discovered by Mantel et al. (2007) that in both mouse and human ES cells the mitotic spindle checkpoint is functional, but fails to induce apoptosis upon cell division arrest. As a result, the cells obtain a

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tetraploid karyotype, which is reduced to the typical near diploid aneuploidity in subsequent divisions. This may facilitate the selection of cells with higher proliferation rate or better propensity for self‐renewal, which may lessen their capacity to differentiate. ES cells lose this ability to survive mitotic arrest upon differentiation, and thus differentiated ES cells are destined to apoptosis upon mitotic checkpoint activation (Mantel et al., 2007). Numerous candidate genes, most of them located on the specific regions of chromosomes 12, 17, and X that are typically associated with culture adaptation were suggested to be involved in the tumorigenicity of the in vitro adapted HESCs. Recent reviews by Harrison et al. (2007) and Baker et al. (2007) inclusively lists important genes on these chromosomal loci, which are suspected to contribute to the in vivo tumorigenicity of culture‐adapted HESCs. One of the most interesting among these genes, located on chromosome 12, is Nanog. It resides on 12p13 (Clark et al., 2004) and is highly expressed in human GCTs and embryonic carcinoma (Hart et al., 2005). Overexpression of Nanog was found to prohibit the differentiation of mouse ES cells under feeder free conditions (Chambers et al., 2003; Mitsui et al., 2003). Similarly, overxpressing Nanog in HESCs allowed them to sustain an undifferentiated phenotype even without feeder cells (Darr et al., 2006). Oct4 is one of the most accurate diagnostic markers in the assessment of the malignancy of human GCTs, as it is specifically expressed in the undifferentiated EC core of teratocarcinomas (Jones et al., 2004). Oct4 was found to work together also with Nanog for the maintenance of pluripotency in ES cells (Wang et al., 2006). Previously, it was suggested that Oct4 itself is a driving force oncogene in mouse GCTs, and that overexpression of it can promote malignancy in mouse ES cells derived tumors (Gidekel et al., 2003). It is currently not known if Nanog and Oct4 are consequently detected in EC cells of teratocarcinomas due to the pluripotency of these cells or whether they are a driving force for tumorigenicity, but it is noteworthy that Nanog and Oct4 are among the few genes found to be required for reprogramming somatic human cells into pluripotent (and tumorigenic) fate (Yu et al., 2007). Another interesting gene is the apoptosis inhibitor, cell cycle regulator survivin. Located on 17q25, survivin is highly expressed in early embryonic tissues, but is virtually absent from adult, terminally differentiated tissues (Adida et al., 1998; Ambrosini et al., 1997; Islam et al., 2000; Li et al., 1998). However, it is also expressed in human testicular GCTs (Weikert et al., 2005) and many other cancers, and is thus seen as a tumorigenic marker. Also on the long arm chromosome 17, the gene BCAS3, recently reported to be expressed in HESCs and in several cancers (Siva et al., 2007), might be implicated in angiogenesis processes of the tumors. Another interesting gene that might be related to the tumorigenicity of HESCs is the human homologue of the mouse ERas. Located on the

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X chromosome, this gene was thought to be a nonfunctional pseudogene called HRasp. However, Takahashi et al. (2003) have found that it is indeed expressed and functional in both mouse and human ES cells. When ERas was added to NIH3T3 fibroblasts, their tumorigenc transformation was promoted. Reciprocally, when mouse ES cells were deprived of ERas, their tumorigenicity was markedly reduced, although they could still form small teratomas. Interestingly, the protein was found to be constitutively active in normal diploid ES cells, but absent from differentiated cells.

D. Tumorigenicity of Nonadapted HESCs In contrast to less mature teratomas generated by culture‐adapted HESCs, nonadapted HESCs were thus far reported to exclusively make mature teratomas. This was determined histologically (Reubinoff et al., 2000), and in some cases by the absence of immunostaining for pluripotency markers like Oct4 (Blum and Benvenisty, 2007; Gertow et al., 2004) and TRA‐1–60, TRA‐1–81, and SSEA4 (Gertow et al., 2004). We have recently reported that BrdU incorporation in established tumors derived from unadapted HESCs demonstrate that within the teratoma all cell types are proliferative, and that no exclusive nests or foci of highly proliferating cells were observed (Blum and Benvenisty, 2007). In the same work we further demonstrated that the tumor as a whole is not clonally derived. Specific differentiated structures within the HESC‐induced teratoma were derived from different cells of origin, thus arguing against clonal selection of a more aggressive HESC clone that takes over the tumor. In contrast, clonality is sometime reported to occur in spontaneous human GCTs (Gillis et al., 1994; Rothe et al., 1999). In a wide ranging survey that was performed by the International Stem Cell Initiative (ISCI) in order to standardize HESC research (Adewumi et al., 2007) many HESC‐induced teratomas were analyzed. It was reported that three of the teratomas actually did include cells that somewhat look like the EC core of teratocarcinoma, but the nature of these EC‐like cells could not be determined, and they were suggested to represent either residual, undifferentiated HESCs, or true embryonic carcinoma cells originated from transformed HESCs. Transplantations of normal, euploid mouse ES cells into adult mice do result sometimes in the formation of tumors that were described as malignant tumors instead of teratomas (Gidekel et al., 2003). Also transplantation of normal mouse embryos gives rise to teratocarcinomas (Solter et al., 1970). The differences observed with human ES cells transplanted to the mouse might be attributed to differences between the transformation of mouse and human cells. Specifically, this may apply that in contrast to mouse ES cells, HESCs are not easily transformed in vivo, perhaps because human

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cells in general are more resilient to transformation than are mouse cells (Rangarajan and Weinberg, 2003; Rangarajan et al., 2004). On the other hand, this difference seen between the tumorigenic capabilities of mouse and human ES cells may be attributed to the difference of species between the transplanted HESCs and the murine environment. It is reasonable to assume that HESC‐derived tumors in the mouse are facing suboptimal conditions regarding growth factors mismatch, difficulties in anastomosing with the host blood system and other discrepancies. Indeed, interspecies host difference in the tumorigenicity of mouse ES cells was demonstrated (Erdo et al., 2003). Erdo co‐workers have transplanted mouse ES cells into the brain of rat and mice hosts. Although mouse ES cells injected to rats hardly resulted in tumor formation, transplanting the same ES cells into mouse brains resulted in the formation of large, very aggressive teratocarcinomas. HESC teratoma experiments in human are obviously not feasible. However, one group has reported on experiments in nonhuman primates’ (i.e., cynomologus macaque) ES cells transplantation back into hosts of the same species (Asano et al., 2003; Shibata et al., 2006). To evade immune rejection in the host, the ES cells were injected in utero, using ultrasound guidance, into the liver or abdominal cavity of monkey fetuses. In these experiments almost all the fetuses developed tumors. The reported tumors were massive, and sometimes killed the host. However, it is not clear whether intraspecies transplantation of primate ES cells results only in benign teratoma or in teratocarcinoma like some of the mouse autologous experiments. Another approach to the question of tumorigenicity of HESCs within human hosts was the injection of HESCs directly into three different human fetal tissues (thymus, lung, and pancreas), which were transplanted earlier in SCID mice (Shih et al., 2007). Surprisingly, it was found that the HESCs transplanted into the human fetal tissues formed aggressive undifferentiated tumors, in which cells still expressed Oct4. In contrast, transplantation of the same cells into the hind leg or under the kidney capsule of the SCID mouse itself resulted in the formation of only mature teratomas. It is noteworthy that the two HESC lines that were used in this experiment displayed normal karyotype before transplantation and also after tumor formation, and did not express CD30. Nonetheless, it still may be that the fetal human tissues used in this experiment were for some unknown reason supportive to HESC self‐renewal (or suppressive for differentiation), similar to other embryonic tissues such as mouse embryonic fibroblasts and human embryonic fibroblasts. It is also interesting that not only species difference but also difference in anatomical location within the same host can be relevant to the tumorigenic outcome; HESCs that were injected into the liver formed tumors that looked more aggressive and less differentiated than those that formed when the same cells were transplanted subcutaneously (Cooke et al., 2006).

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More provocatively, when HESCs were transplanted into the brain ventricle of neonatal mice, they differentiated to functional neurons and integrated into the host brain without teratoma formation (Muotri et al., 2005). This may be explained by the presence of an environment rich in developmental signals, thus directing the HESCs to take part in the surrounding tissue. The tumorigenic feature of HESCs may perhaps be explained by the fact that ES cells already normally possess some of the features considered necessary for tumor formation, such as immortality, telomerase expression, resistance to contact inhibition, and abrogated cell cycle. However, there is still much to discover regarding this unique phenomenon.

IV. HESC‐INDUCED TERATOMAS AS A MODEL FOR EARLY HUMAN DEVELOPMENT In line with the notion that normal HESCs can differentiate to form teratoma‐like tumors without being transformed, HESC‐derived tumors are seen by some not as a tumor at all, but rather as failed progress of normal embryonic development, due to the incorrect localization of the developing cells (Lensch and Ince, 2007; Lensch et al., 2007). In that context, this tumorigenic activity of HESCs can function as a very promising tool by which one can study the very early stages of human embryonic development. These are actually inaccessible to research, due to the unavailability of normal human embryos at theses stages, and the obvious ethical restriction regarding human experimentation. Moreover, as the early developmental stages of mouse and human embryos differ in many ways (Dvash and Benvenisty, 2004), the use of HESCs, as a tool for developmental studies, both of normal development and as a model for developmental diseases, is sometimes the only possibility. This is further stressed by the fact that in vitro differentiation of HESCs fails to reach the level of tissue complexity seen upon in vivo differentiation (Blum and Benvenisty, 2005).

A. Modeling Normal Embryogenesis Gertow et al. (2004) were the first to try and describe in detail the development of various tissues within HESC‐induced teratoma using systematic histological examinations and a large set of antibodies recognizing differentiated tissues. They report that within the teratoma, tissue types known to be inductive of each other were in close proximity. Furthermore, they observed that the degree of differentiation within the teratoma is higher in tumors that were grown for a longer time in the mouse. Intertissue induction was also

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suggested from the observation by Lavon et al. (2004), who noticed that hepatic‐like cells were localized adjacent to cardiomyocytes in HESC‐induced teratomas. These observations suggested that the teratoma tissue is developing in a way that resembles normal events in the developing embryo. In order to assess this issue experimentally, we have performed experiments in which distinct differentiated structures were excised (using laser capture microdissection) from teratomas that were generated from a combination of three different HESC lines, and proved that they are formed by the assembly of cells from different origins, meaning that the teratoma environment is indeed inductive (Blum and Benvenisty, 2007). In contrast, Gerecht‐Nir et al. (2004), studying vasculogenesis in HESC‐ induced teratomas, have found that most of the blood vessels within the tumor originate from the murine host. They have stated that vascular development within HESC‐induced teratomas does not completely mimic normal human development and that, at least in this case, in vitro models might prove more useful.

B. Modeling Genetic Diseases Eiges et al. (2007) have recently isolated HESCs from embryos carrying the fragile X syndrome. These cells were used to study the silencing of FMR1. This model is very relevant, since mouse model fail to recapitulate the inactivation of FMR1 that occurs in the fragile X patients. Here, the use of teratomas induced from the mutated HESCs was instrumental in the study of the developmental silencing of FMR1 in neuronal cells. Recently, teratomas from mouse ES cells carrying an additional copy of human chromosome 21 were reported (Mensah et al., 2007). The study of such teratomas was aimed at understanding the means by which this specific trisomy affects neuronal differentiation in Down syndrome patients. Histological and immunohistochemical examination of these teratomas show less neuronal differentiation in the affected teratomas than in the parental cell line. However, a more convincing proof of this phenomenon in human Down syndrome will be to compare neuronal differentiation in teratomas from human embryos with Down syndrome that will be identified through PGD.

C. Utilizing HESC‐Induced Teratomas as a Surrogate Human Environment for Cancer Research Another creative use of HESC‐induced teratomas has been their use as a tool to study the relationship between normal human tissues and cancer development. These relationships are very difficult to study using

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conventional mouse models. Thus, Tzukerman et al. (2003, 2006) established human teratomas in immunodeficient mice hosts, and then injected genetically labeled human cancer cells directly into the teratoma. By tracing the behavior of the caner cells within the teratomas, they extrapolated conclusions on the alleged behavior of these malignant cells in human environment.

V. HESC‐INDUCED TERATOMAS AS A CLINICAL HURDLE There are still several obstacles on the way to the implementation of HESCs in cellular therapy, the most prominent being immune rejection and the tumorigenicity of HESCs‐based tissues (Parson, 2006; Vogel, 2005). Recently, the issue of immune rejection of HESC‐based grafts has made enormous progress towards resolution with the report of two different approaches to generating patient‐specific pluripotent stem cells. First, Byrne et al. (2007) have succeeded in the cloning of a nonhuman primate, a task that has been considered technically impossible (Simerly et al., 2003). Second, Takahashi et al. (2007) and Yu et al. (2007) have independently reported the successful reprogramming of somatic human cells into pluripotent cells perfectly resembling HESCs. These scientific advancements have brought the concern regarding the tumorigenicity of the implanted cells into the prime spotlight. The formation of a teratoma as a clinical outcome of HESC transplantation in human patients is completely unacceptable, regardless of its perception as a less‐differentiated tumor from culture‐adapted cells or as a disorganized bulk of normal embryonic tissues. Accordingly, several strategies have been implemented in order to tackle the dangerous tumorigenic potential of HESC‐ induced transplants (Fig. 3). These have been thoroughly reviewed by Hentze et al. (2007), who comprehensively summarize and categorize the clinical hurdles facing utilization of HESC‐based grafts in the clinic. We will give general examples of approaches designed to evade teratoma formation in grafted HESCs.

A. General Ablation of Teratoma Cells To be able to control and ablate the transplanted cells, if a teratoma forms after grafting, Schuldiner et al. (2003) have genetically engineered a HESC line to carry as a transgene the viral thynidine kinase (HSV‐tk) gene. Upon treatment with ganciclovir, an antiviral drug designed to induce apoptosis in the presence of herpes simplex thynidine kinase, the cells carrying this so‐called “suicide gene” are eliminated. Using this system, Schuldiner et al.

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Terminal differentiation/eliminating residual pluripotent cells

Pre-transplantation

Interfering with tumor progression genes

Tumor formation

Suicide genes

Tumor detection

Fig. 3 Strategies for preventing teratoma formation from HESC‐based grafts. (A) The cell culture can be terminally differentiated prior to transplantation. Residual tumorigenic cells can be excluded from the transplant using various sorting techniques. (B) Different mechanisms that are involved in teratoma formation can be targeted to produce HESCs which are intrinsically unable to generate tumors after transplantation. (C) Cells carrying a “suicide genes” can be eliminated after teratoma formation using specific drugs.

were able to significantly eradicate in vivo established tumors in mice burdening HESC‐induced teratomas. This pioneering approach with human ES cells was recently repeated with slight modifications using mouse ES cells. Thus, Cao et al. (2006) infected mouse ES cells with a lentivirus carrying a triple fusion transgene incorporating firefly luciferase, monomeric red fluorescence protein, and a truncated thymidine kinase. This enabled them to follow the kinetics of teratoma formation after injection of the cells into the myocardium of immunosuppressed rats. With this system, no tumors were created when the rats were treated with ganciclovir. In yet another report, Jung et al. infected mouse ES cells with a lentivirus carrying TK and green fluorescence protein. Again, they were able to completely eliminate teratomas arising from transplantation of the modified ES cells to the flank and into the CNS of SCID mice after treatment with ganciclovir (Jung et al., 2007). One drawback of this method is that by applying ganciclovir, all proliferating cells carrying the suicide gene are eliminated, thus while killing the tumor the normal differentiating cells will also be affected. Another drawback is that genetically modified cells are used, increasing their chance for transformation.

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B. Differentiation to Eliminate Tumorigenic Cells As discussed earlier, neither euploid nor culture‐adapted HESCs generate teratomas after being terminally differentiated prior to transplantation. Hence, a large number of efforts are being made to completely differentiate HESCs before grafting them, and to eliminate residual pluripotent cells that may hide in the culture. This is especially important giving the fact that a work on mouse ES cells aimed at finding the minimal amount of ES cells sufficient to form a teratoma revealed that even single pluripotent ES cells are able to generate this tumor (Lawrenz et al., 2004). Most of the approaches thus far tested to prevent the formation of teratomas from both mouse and human ES cells were aimed at terminally differentiating them in order to get rid of the pluripotent tumorigenic population, but not all were reported successful. For example, Leor et al. (2007) have transplanted small pieces of HESCs derived beating cardiomyocytes into the heart of athymic nude rats. They report that the injected cells did not integrate fully into the infracted myocardium, but differentiated to various types of fibrotic and myofibrotoc tissues and a teratoma occurred in one of the animals. Similarly, Fujikawa et al. (2005) report on the development of a teratoma after transplantation of allegedly insulin‐secreting cells. Here, mouse ES cells were differentiated in vitro and were shown to express insulin mRNA as well as C peptide, and were able to complement hyperglycemia in the host for 3 weeks. Again, incomplete differentiation has led to the formation of a teratoma and to the end of the experiment. On the other hand, other methods of differentiation have proven more successful. Interestingly, these were usually designated to produce neurons, suggesting the possibility that the brain is either less permissive on teratoma formation (maybe owing to a more inductive environment), or that neurons are easier to differentiate purely. Two examples are works of Reubinoff et al. (2001) and Zhang et al. (2001), who succeeded, by different methods composed of a complex succession of steps, to generate neural precursors that did intercalate into mice brains, but did not create teratomas.

C. Sorting for Nontumorigenic Populations or against Pluripotent Cells Another approach is to specifically sort out residual pluripotent tumorigenic HESCs after differentiation, or sort for a desired progenitor population. Several years ago, a fluorescent marker under the regulation of a pluripotent marker (Rex‐1) was introduced into HESCs (Eiges et al., 2001). These new cell lines were fluorescent as undifferentiated cells and

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lost their fluorescence upon differentiation. It was shown that the undifferentiated tumorigenic cells can be sorted out from a mixed population of HESCs and their differentiated progenies. Another effort to specifically ablate pluripotent cells after transplantation without genetic manipulation has been the use of ceramide analogues that specifically induce apoptosis in pluripotent cells (Bieberich et al., 2004). It was discovered that the expression of the PAR‐4 gene, whose encoded protein is involved in the apoptotic response of ES cells to ceramides during neural differentiation, is colocalized with the expression of the OCT4 gene in differentiating ES cells. By incubation of the differentiating culture with a specific ceramide analogue, residual pluripotent cells were indeed eliminated, resulting in no teratoma formation after transplantation of these cultures into mice. Another example is the work by Shibata et al. (2006). Here, cynomolgus monkey ES cells differentiated into hematopoietic cells generated teratoma upon in vivo transplantation into embryos of the same species. Sorting this population for SSEA4‐negative cells by means of flow cytometry resulted in a tumorigenic‐free population. Alternatively, sorting the differentiated population according to a tissue‐ specific marker can provide highly purified populations of nontumorigenic cells. Fukuda et al. (2006) and Chung et al. (2006) used FACS to purify neural progenitors for sox1GFP‐positive cells. These sox1‐positive cells engrafted well and did not form tumors, in contrast to the sox1GFP‐negative fraction, which was tumorigenic. Similarly, Barberi et al. (2007) succeeded in generating a pure population of nontomorigenic myoblasts by sorting for CD73þ/NCAMþ cells.

VI. CONCLUDING REMARKS Blastocyst‐derived ES cells are the in vitro counterparts of the malignant EC cells found in spontaneous teratocarcinomas. The study of HESCs has emerged from pivotal experiments and observations on teratocarcinoma and teratoma GCTs, and is now coming of age. Being the in vitro successors of the pluripotent ICM cells, it is hypothesized that the differentiation of nonadapted HESCs in vivo resembles normal embryonic processes, albeit in a disorganized manner. It is thus attractive to use HESC‐induced teratomas to study development in humans, as early stages of human embryo, especially around the time of implantation, are inaccessible. This is also true in the case of developmental disorders, where an animal model is sometimes insufficient. HESCs can acquire karyotypic changes in culture upon stressful prolonged growth. These culture‐adapted HESCs then resemble, karyotypically, malignant EC cells. Specific genes like Nanog and ERas, some of which are

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reported to relate to cancer, are located on the chromosomes that are involved in this culture adaptation, and the adapted cells are suggested to generate less mature tumors. Taken together, the reports regarding the outcome of the transplantation of naı¨ve or culture‐adapted HESCs that were surveyed here indicate that there may actually be different levels of tumorigenicity displayed by HESCs. Thus, as naı¨ve, unadapted cells generate only mature teratomas, the tumorigenicity of culture‐adapted HESCs is less mature, and is suggested to be more aggressive. The ability of HESCs to differentiate has led to high expectations regarding the use of them in medicine. These are somewhat clouded by HESCs notorious habit to generate teratomas. Any of the techniques thus far described is not sufficient to completely preclude the formation HESC‐induced teratoma in human patients. However, methods that will aim against tumorigenic genes that are normally expressed in unadapted HESCs may prevent this process. Clinical translation of HESCs cannot tolerate any type of tumorigenicity of HESC‐based grafts. In view of the enormous progress in the field of regenerative medicine following the soon expected ability to generate patient‐specific pluripotent stem cells, there is an urgent need for more experiments that will solve the problem of HESC‐induced tumorigenicity.

ACNOWLEDGMENTS We thank Yoav Mayshar and Rina Klinov for critically reading the manuscript and Tamar Golan‐Lev for assistance with preparation of the figures. This work was partially supported by funds from Bereshit Consortium, the Israeli Ministry of Trade and Industry (Grant number 37675), and by the European Community (ESTOOLS, Grant number 018739).

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