PURPOSE: In previous studies, we have used human embryonic stem cells (hESC) to generate a tissue microenvironment in immunocompromised mice as an experimental approach for studying human tumorigenesis. We now examine the attributes of such a cellular microenvironment in supporting the growth of human cancer cells freshly harvested from malignant ovarian ascites and to determine whether there are differences among subsets of ascites-derived cancer cells in terms of tumorigenic capacity in the conventional murine xenograft model and in the hESC-derived microenvironment. EXPERIMENTAL DESIGN: Freshly harvested malignant ovarian ascites-derived cancer cells and six derivative ovarian cancer cell subpopulations (CCSP) were characterized for ovarian cancer-associated biomarker expression both in vitro and in vivo and for their capacity to generate tumors in the two models. RESULTS: Ovarian cancer-associated biomarkers were detected in the ascites-derived cancer cells and in the six newly established CCSPs. Nevertheless, certain CCSPs that did not develop into tumors in a conventional murine xenograft model did generate tumors in the hESC-derived cellular microenvironment, indicating variable niche dependency for the tumorigenic capacity of the different CCSPs. The hESC-derived microenvironment provided an improved niche for supporting growth of certain tumor cell subpopulations. CONCLUSIONS: The results highlight the experimental utility of the hESC-derived cellular microenvironment to enable functional distinction of CCSPs, including the identification of cells that do not grow into a tumor in the conventional direct tumor xenograft platform, thereby rendering such cells accessible to characterization and testing of anticancer therapies.

Human embryonic stem cells (hESC) are pluripotent lines that can differentiate in vitro into cell derivatives of all three germ layers, including cardiomyocytes. Successful application of these unique cells in the areas of cardiovascular research and regenerative medicine has been hampered by difficulties in identifying and selecting specific cardiac progenitor cells from the mixed population of differentiating cells. We report the generation of stable transgenic hESC lines, using lentiviral vectors, and single-cell clones that express a reporter gene (eGFP) under the transcriptional control of a cardiac-specific promoter (the human myosin light chain-2V promoter). Our results demonstrate the appearance of eGFP-expressing cells during the differentiation of the hESC as embryoid bodies (EBs) that can be identified and sorted using FACS (purity>95%, viability>85%). The eGFP-expressing cells were stained positively for cardiac-specific proteins (>93%), expressed cardiac-specific genes, displayed cardiac-specific action-potentials, and could form stable myocardial cell grafts following in vivo cell transplantation. The generation of these transgenic hESC lines may be used to identify and study early cardiac precursors for developmental studies, to robustly quantify the extent of cardiomyocyte differentiation, to label the cells for in vivo grafting, and to allow derivation of purified cell populations of cardiomyocytes for future myocardial cell therapy strategies.-- Huber, I., Itzhaki, I., Caspi, O., Arbel, G., Tzukerman, M., Gepstein, A., Habib, M., Yankelson, L., Kehat, I., Gepstein, L. Identification and selection of cardiomyocytes during human embryonic stem cell differentiation.

Human embryonic stem cells (hESC) are pluripotent lines that can differentiate in vitro into cell derivatives of all three germ layers, including cardiomyocytes. Successful application of these unique cells in the areas of cardiovascular research and regenerative medicine has been hampered by difficulties in identifying and selecting specific cardiac progenitor cells from the mixed population of differentiating cells. We report the generation of stable transgenic hESC lines, using lentiviral vectors, and single-cell clones that express a reporter gene (eGFP) under the transcriptional control of a cardiac-specific promoter (the human myosin light chain-2V promoter). Our results demonstrate the appearance of eGFP-expressing cells during the differentiation of the hESC as embryoid bodies (EBs) that can be identified and sorted using FACS (purity>95%, viability>85%). The eGFP-expressing cells were stained positively for cardiac-specific proteins (>93%), expressed cardiac-specific genes, displayed cardiac-specific action-potentials, and could form stable myocardial cell grafts following in vivo cell transplantation. The generation of these transgenic hESC lines may be used to identify and study early cardiac precursors for developmental studies, to robustly quantify the extent of cardiomyocyte differentiation, to label the cells for in vivo grafting, and to allow derivation of purified cell populations of cardiomyocytes for future myocardial cell therapy strategies.-- Huber, I., Itzhaki, I., Caspi, O., Arbel, G., Tzukerman, M., Gepstein, A., Habib, M., Yankelson, L., Kehat, I., Gepstein, L. Identification and selection of cardiomyocytes during human embryonic stem cell differentiation.

There is currently no available experimental system wherein human cancer cells can be grown in the context of a mixed population of normal differentiated human cells for testing biological aspects of cancer cell growth (e.g., tumor cell invasion and angiogenesis) or response to anti-cancer therapies. When implanted into immunocompromised mice, human embryonic stem cells develop teratomas containing complex structures comprising differentiated cell types representing the major germ line-derived lineages. We sought to determine whether human cancer cells would grow within such teratomas and display properties associated with malignancy, such as invasiveness and recruitment of blood vessels. HEY ovarian cancer cells stably expressing an H2A-GFP fusion protein (HEY-GFP) injected into mature teratomas developed into tumors, which allowed tracking of tumor cell invasion and recruitment of human teratoma-derived blood vessels. This provides a straightforward and powerful approach to studying the biological properties of cancer cells within the microenvironment of normal differentiated human cells.

Inactivation of p53 and activation of telomerase occur in the majority of human cancers, raising the possibility of a link between these two pathways. Overexpression of wild-type p53 down-regulates the enzymatic activity of telomerase in various cancer cell lines through transcriptional repression of its catalytic subunit, human telomerase reverse transcriptase (hTERT). In this study, we re-evaluated the role of p53 in telomerase regulation using isogenic cell lines expressing physiological levels of p53. We demonstrate that endogenous wild-type p53 was able to down-regulate telomerase activity, hTERT mRNA levels, and promoter activity; however, the ability to repress hTERT expression was found to be cell type-specific. The integrity of the DNA-binding core domain, the N-terminal transactivation domain, and the C-terminal oligomerization domains of p53 was essential for hTERT promoter repression, whereas the proline-rich domain and the extreme C terminus were not required. Southwestern and chromatin immunoprecipitation experiments demonstrated lack of p53 binding to the hTERT promoter, raising the possibility of an indirect repressive mechanism. The down-regulation of hTERT promoter activity was abolished by a dominant-negative E2F1 mutant. Mutational analysis identified a specific E2F site responsible for p53-mediated repression. Knockdown of the key p53 transcriptional target, p21, was sufficient to eliminate the p53-dependent repression of hTERT. Inactivation of the Rb family using either viral oncoproteins or RNA interference attenuated the repression. Inhibition of histone deacetylases also interfered with the repression of hTERT by p53. Therefore, our results suggest that repression of hTERT by endogenous p53 is mediated by p21 and E2F.

The awareness of the important role that the surrounding tissue microenvironment and stromal response play in the process of tumorigenesis has grown as a result of in vivo models of tumor xenograft growth in immunocompromised mice. In the current study, we used human embryonic stem cells in order to study the interactions of tumor cells with the surrounding microenvironment of differentiated human cell tissues and structures. Several cancer cell types stably expressing an H2A-green fluorescence protein fusion protein, which allowed tracking of tumor cells, were injected into mature teratomas and developed into tumors. The salient findings were:(a) the observation of growth of tumor cells with high proliferative capacity within the differentiated microenvironment of the teratoma,(b) the identification of invasion by tumor cells into surrounding differentiated teratoma structures, and (c) the identification of blood vessels of human teratoma origin, growing adjacent to and within the cancer cell-derived tumor. Mouse embryonic stem cell-derived teratomas also supported cancer cell growth, but provided a less suitable model for human tumorigenesis studies. Anticancer immunotherapy treatment directed against A431 epidermoid carcinoma cell-related epitopes induced the complete regression of A431-derived tumor xenografts following direct i.m. injection in immunocompromised mice, as opposed to corresponding tumors growing within a human embryonic stem cell-derived microenvironment, wherein remnant foci of viable tumor cells were detected and resulted in tumor recurrence. We propose using this novel experimental model as a preclinical platform for investigating and manipulating the stromal response in tumor cell growth as an additional tool in cancer research.

Epigenetics refers to the durable changes affecting the genome of an individual during development and aging, but which are not necessarily passed on to subsequent generations. Among the best studied of these epigenetic changes is the shortening of chromosome ends or telomeres. Telomeres are specialized structures, consisting of characteristic DNA repeat sequences and the complex of associated proteins, which cap and protect chromosome ends and serve to preserve genome integrity. In most somatic cells, progressive rounds of cell division are associated with telomere shortening. Such progressive attrition of telomere length eventuates in loss of replicative capacity (cellular senescence). In order to protect the germline and the subpopulation of stem cells from senescence, mechanisms have evolved to prevent telomere attrition in these cellular compartments. The most common and best studied mechanism involves the activation of a ribonucleoprotein enzyme complex, known as telomerase. Activity of telomerase circumvents loss of replicative capacity, by preserving telomere length and chromosome integrity. Hence the detailed mechanisms governing the expression and activity of telomerase have been intensively studied in development and differentiation. Early embryonic development and cellular differentiation are associated with a progressive diminution in telomerase activity. This decrease in activity is principally mediated at the level of the promoter for the gene encoding the catalytic unit of the telomerase complex. Unraveling the detailed mechanisms involved in the regulation of telomere length and telomerase activity will have important and far-reaching implications in understanding many aspects of human health and disease, ranging from accelerated aging syndromes to cancer pathogenesis, among others. Furthermore, insights gleaned from continuing research in this area will likely be applicable to the development of strategies to circumvent cellular senescence in regenerative medicine and stem cell therapeutics in the years to come.

The recent characterization of MHC-displayed tumor-associated antigensthat recognize effector cells of the immune system has created new perspectives for cancer therapy. Antibodies that recognize these tumor-associated MHC-peptide complexes with the same specificity as the T-cell antigen receptor will therefore be valuable tools for immunotherapy as well as for studying antigen presentation in human cancers. Most tumor-associated antigens are expressed in only one or a few tumor types; however, recently specific T-cell epitopes derived from the telomerase catalytic subunit (hTERT) that are widely expressed in many cancers were identified and shown to be recognized by CTLs derived from cancer patients. We selected a large nonimmune repertoire of phage Fab antibodies on recombinant human class I HLA-A2 complexes displaying two distinct antigenic T-cell epitopes derived from hTERT. We isolated a surprisingly large panel of high-affinity human recombinant Fab antibodies that exhibited peptide-specific, MHC-restricted binding characteristics of T cells. The analyzed Fabs not only recognize the cognate MHC-peptide complex in a recombinant soluble form but also the native complex as displayed on the surface of antigen-presenting cells and hTERT-expressing tumor cells. These findings demonstrate for the first time the ability to transform the unique fine specificity but low intrinsic affinity of TCRs on T cells into high-affinity soluble antibody molecules endowed with a T-cell antigen receptor-like specificity. These molecules may prove to be very important and widely applicable for monitoring the expression of specific MHC-peptide complexes on the surface of tumor and immune cells, for structure-function studies of TCR-peptide-MHC interactions, as well as for developing new targeting agents for immunotherapy.