Search

The potential for the formation of teratomas or other neoplasms is a major safety roadblock to clinical application of pluripotent stem cell therapies. Preclinical assessment of the risk of tumor formation in this context poses considerable scientific and regulatory challenges, especially because animal xenograft models may not properly reflect the long-term tumorigenic potential of human cells. A better understanding of the biology of spontaneously occurring teratomas and related tumors in humans can help to guide efforts to assess and minimize the potential hazards of embryonic stem cell or induced pluripotent stem cell therapeutics. Here we review the features of teratomas derived experimentally from human pluripotent stem cells and argue that they most closely resemble spontaneous benign teratomas that occur early in both mouse and human life. The natural history and pathology of these spontaneously occurring teratomas provide important clues for preclinical safety assessment and patient monitoring in trials of stem cell therapies.

We describe the cloning and initial characterization of a novel cDNA from human embryonal carcinoma (EC) cells. This cDNA, which we named human growth differentiation factor 3 (hGDF3), encodes the homologue of mouse GDF3, a TGFbeta superfamily member belonging to the Growth/Differentiation Factors. We have analysed the expression of hGDF3 in human embryonal carcinoma cell lines and in primary testicular germ cell tumours of adolescents and adults (TGCTs). Expression of hGDF3 in human EC cell lines is stem cell-specific, is down-regulated upon RA-mediated differentiation and is increased upon culture of the cells in the presence of activin A. In TGCTs, hGDF3 expression is low in seminomas, while expression in non-seminomas is readily detectable and appears to be associated with the EC and yolk sac components in the tumours. We have also mapped the hGDF3 locus to the short arm of human chromosome 12, a region consistently overrepresented in human testicular germ cell tumours. Thus, hGDF3 represents an embryonal carcinoma stem cell-associated marker both in vitro and in vivo.

Like the oocyte, the cells of the early embryo, and primordial germ cells, human teratocarcinoma stem cells are pluripotent, capable of giving rise to a wide range of somatic and extraembryonic tissues. Growth factors which regulate the growth of multipotent stem cells in the mouse have been identified, but none of these have been shown conclusively to have similar effects on human or primate multipotent stem cells. CD30 is a member of the tumour necrosis factor receptor superfamily with a restricted pattern of tissue distribution, limited to immune cells, decidual tissue, and human embryonal carcinoma: in common with other embryonal carcinoma markers, CD30 is found in foci of cells in a sub-population of seminomas. CD30 ligand is a transmembrane protein, structurally related to tumour necrosis superfamily members TNF alpha, TNF beta, and CD40. CD30 ligand is expressed by T and B lymphocytes, macrophages, and a variety of normal haematopoietic cells and tumours derived from them, and exerts pleiotropic effects on normal and malignant lymphoid cells, including death, differentiation, or cell division. Studies on cultured cell lines derived from human embryonal carcinomas and yolk sac carcinomas confirm CD30 expression in the former but not the latter, and show that CD30 expression is down-regulated during stem cell differentiation in vitro. Transcripts for CD30 ligand are found at highest levels in yolk sac carcinoma cell lines, but are also found in embryonal carcinoma. CD30 ligand protein is detected in yolk sac carcinoma and nullipotent embryonal carcinoma cell lines. Exogenous CD30 ligand has no effect on multipotent human stem cell growth in vitro. However, the receptor-ligand pair may function in autocrine regulation of embryonal carcinoma stem cells. CD30 and its ligand are candidate stem cell identity factors, juxtacrine regulators whose sole function is to identify a cell's position in a developmental hierarchy.

Cultured cell lines from human teratomas provide models to study molecules which might regulate tumour growth or normal embryonic development. Here, we describe efforts to delineate the role of transcription factors, extracellular matrix, and growth factors in controlling growth and differentiation of human embryonal carcinoma and yolk sac tumour.

Human embryonal carcinoma cells sometimes display the developmental potential of early embryonic stem cells. While available data do not clearly identify a counterpart of these tumor cells in normal development, previous comparisons of human embryonal carcinoma and yolk sac carcinomas indicated that these cell types are closely related, and suggested that embryonal carcinoma cells might resemble the progenitors of extraembryonic endoderm. To analyse further cell-differentiation lineage in these tumors, we produced monoclonal antibodies to cytostructurally associated antigens of human embryonal carcinoma cells. Spleen cells from mice immunized with a detergent-insoluble extract of cultured human embryonal carcinoma cells were fused to NS-1 myeloma cells, and hybridoma supernatants were screened by indirect immunofluorescence on the immunizing cell line, then on a panel of cell lines derived from human embryonal carcinomas, yolk sac carcinomas, and a range of neoplastic and normal tissues. Monoclonal antibody GCTM-1 stained the nuclei of all human cells tested and served as a positive control; this antibody immunoprecipitated proteins of 85 and 66 k Da from human embryonal carcinoma cells. GCTM-2 recognized an epitope on a 200-k Da extracellular protein present on the surface of embryonal carcinoma cells, and stained the surface of visceral yolk sac-type carcinoma and colorectal carcinoma cells as well. Enzymatic analysis of carbohydrate residues on the GCTM-2 antigen revealed that it was a keratan sulphate proteoglycan, and suggested that the epitope recognized by the antibody lies on the core protein. In immunoblots, antibody GCTM-3 bound to a 57-k Da cytoskeletal protein expressed in human embryonal carcinoma. This antibody decorated filamentous arrays in cell lines from human embryonal carcinoma, visceral yolk sac carcinoma, parietal yolk sac carcinoma (endodermal sinus tumour), and adenocarcinoma and large cell carcinoma of the lung. Antibody GCTM-4 recognized a determinant present on a 69-k Da polypeptide, associated with a component of the lysosomal compartment, which was expressed in embryonal carcinoma cells, but no other cell type tested. The results with this antibody panel thus allow distinction between human embryonal carcinoma and yolk sac carcinoma, but provide further evidence of a close relationship between these cell types.

The understanding of differentiation in human teratomas requires a better definition of the phenotype and developmental potential of the stem cells in these tumours. We describe the characterization of 6 new cell lines from human testicular teratomas which are representative of 3 distinct cell types. Cell lines GCT 27, GCT 35, and GCT 48, identified as embryonal carcinoma, comprise epithelial cells which express cytokeratin intermediate filaments and desmoplakins, as determined by indirect immunofluorescence microscopy. A minority of the cells also express vimentin. Most cells in these cultures show surface staining with monoclonal antibodies (MAbs) to stage-specific embryonic antigens SSEA 3 and SSEA 4 but not SSEA 1. Staining with MAb W6/32, which recognizes HLA A,B and C chains in the presence of beta-2 microglobulin, is not above background level. When injected into nude mice, GCT 27 cells form tumours consisting of embryonal carcinoma, somatic tissues, and cells positive in immunocytochemical assays for alphafoetoprotein (AFP) and human chorionic gonadotrophin (HCG); GCT 35 cells form embryonal carcinomas with foci of AFP and HCG-positive cells; and GCT 48 cells form embryonal carcinoma only. A second type of cell (GCT 72) displays some properties of rodent visceral endoderm. GCT 72 cells contain cytokeratin intermediate filaments, but not vimentin, and show very strong staining at cell borders with anti-desmoplakin I + II antibody. At the cell surface, GCT 72 cells express the epitopes recognized by antibodies to SSEA 3, SSEA 4, and SSEA 1; staining with W6/32 is negligible. Levels of AFP in supernatants from GCT 72 cultures are in excess of 500 KIU/I. The tumours formed following inoculation of nude mice with GCT 72 cells are solid yolk-sac tumours, with all cells strongly positive for AFP. A third cell type (GCT 44 and GCT 46), resembles in some ways rodent parietal endoderm. These cells uniformly coexpress keratin and vimentin intermediate filaments, but not desmoplakins. The determinants recognized by MAbs to SSEA 3, 4, or 1 are not detected on the majority of cells in these cultures. In striking contrast to the other teratoma lines, these cells can attach to untreated tissue culture plastic in serum-free medium and may be serially cultivated under these conditions. The tumours formed in nude mice by these 2 cell lines are yolk-sac carcinomas with endodermal sinus tumour histology. Thus, human testicular teratomas consist of epithelial cells which may be nullipotent, pluripotent or committed to extraembryonic endodermal differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Clinical and experimental data indicate that human embryonal carcinoma cells are unusually sensitive to the antitumor drug cis-diamminedichloroplatinum(II) (cisplatin), but the basis of this sensitivity is unknown. Using colony formation assays we measured survival of cultured human embryonal carcinoma cells following cisplatin treatment and related survival to the amount of platinum bound to DNA, determined by isolation of cellular DNA and flameless atomic absorption spectrometry, over a range of drug doses. Similar measurements were carried out on F9 mouse embryonal carcinoma cells and on a fibroblast cell strain from a patient with the genetic disease Fanconi's anemia, a syndrome associated with hypersensitivity to cytotoxic and clastogenic effects of difunctional DNA-binding agents. These results were compared with similar analyses on a variety of cultured cells from previous studies. The embryonal carcinoma cells and the Fanconi's anemia fibroblast strain were among the most sensitive cells on a dose-response basis. Since the amount of platinum bound to DNA after treatment of these cells was similar to values reported for many other cell types, it follows that mouse and human embryonal carcinoma cells are inherently sensitive to DNA-bound platinum adducts, to a degree approaching the sensitivity of fibroblasts from patients with xeroderma pigmentosum and Fanconi's anemia.