Evaluating the genomic and sequence integrity of human ES cell lines; comparison to normal genomes.

Copy number variation (CNV) is a common chromosomal alteration that can occur during in vitro cultivation of human cells and can be accompanied by the accumulation of mutations in coding region sequences. We describe here a systematic application of current molecular technologies to provide a detailed understanding of genomic and sequence profiles of human embryonic stem cell (hESC) lines that were derived under GMP-compliant conditions. We first examined the overall chromosomal integrity using cytogenetic techniques to determine chromosome count, and to detect the presence of cytogenetically aberrant cells in the culture (mosaicism). Assays of copy number variation, using both microarray and sequence-based analyses, provide a detailed view genomic variation in these lines and shows that in early passage cultures of these lines, the size range and distribution of CNVs are entirely consistent with those seen in the genomes of normal individuals. Similarly, genome sequencing shows variation within these lines that is completely within the range seen in normal genomes. Important gene classes, such as tumor suppressors and genetic disease genes, do not display overtly disruptive mutations that could affect the overall safety of cell-based therapeutics. Complete sequence also allows the analysis of important transplantation antigens, such as ABO and HLA types. The combined application of cytogenetic and molecular technologies provides a detailed understanding of genomic and sequence profiles of GMP produced ES lines for potential use as therapeutic agents.

Chromosome 7 and 19 trisomy in cultured human neural progenitor cells.

BACKGROUND: Stem cell expansion and differentiation is the foundation of emerging cell therapy technologies. The potential applications of human neural progenitor cells (hNPCs) are wide ranging, but a normal cytogenetic profile is important to avoid the risk of tumor formation in clinical trials. FDA approved clinical trials are being planned and conducted for hNPC transplantation into the brain or spinal cord for various neurodegenerative disorders. Although human embryonic stem cells (hESCs) are known to show recurrent chromosomal abnormalities involving 12 and 17, no studies have revealed chromosomal abnormalities in cultured hNPCs. Therefore, we investigated frequently occurring chromosomal abnormalities in 21 independent fetal-derived hNPC lines and the possible mechanisms triggering such aberrations. METHODS AND FINDINGS: While most hNPC lines were karyotypically normal, G-band karyotyping and fluorescent in situ hybridization (FISH) analyses revealed the emergence of trisomy 7 (hNPC(+7)) and trisomy 19 (hNPC(+19)), in 24% and 5% of the lines, respectively. Once detected, subsequent passaging revealed emerging dominance of trisomy hNPCs. DNA microarray and immunoblotting analyses demonstrate epidermal growth factor receptor (EGFR) overexpression in hNPC(+7) and hNPC(+19) cells. We observed greater levels of telomerase (hTERT), increased proliferation (Ki67), survival (TUNEL), and neurogenesis (beta(III)-tubulin) in hNPC(+7) and hNPC(+19), using respective immunocytochemical markers. However, the trisomy lines underwent replicative senescence after 50-60 population doublings and never showed neoplastic changes. Although hNPC(+7) and hNPC(+19) survived better after xenotransplantation into the rat striatum, they did not form malignant tumors. Finally, EGF deprivation triggered a selection of trisomy 7 cells in a diploid hNPC line. CONCLUSIONS: We report that hNPCs are susceptible to accumulation of chromosome 7 and 19 trisomy in long-term cell culture. These results suggest that micro-environmental cues are powerful factors in the selection of specific hNPC aneuploidies, with trisomy of chromosome 7 being the most common. Given that a number of stem cell based clinical trials are being conducted or planned in USA and a recent report in PLoS Medicine showing the dangers of grafting an inordinate number of cells, these data substantiate the need for careful cytogenetic evaluation of hNPCs (fetal or hESC-derived) before their use in clinical or basic science applications.

Evaluation of a breast/ovarian cancer genetics referral screening tool in a mammography population.

PURPOSE: Simple screening tools are needed to facilitate the appropriate referral of patients for genetic counseling and testing for BRCA1/2 mutations. This study evaluated the reliability and accuracy of a "referral screening tool" designed for rapid identification of individuals at potential hereditary risk for breast/ovarian cancer. METHODS: The referral screening tool was administered to 2464 unselected women undergoing screening mammography. Detailed four-generation cancer pedigrees were collected by telephone interview on a random subset of 296 women. The pedigrees were analyzed using four established hereditary risk models (BRCAPRO, Myriad II, BOADICEA, FHAT), with a > or =10% BRCA1/2 mutation probability or a FHAT score of > or =10 as the definition of "high risk." RESULTS: The referral screening tool identified 6.2% of subjects as screen "positive" (high risk). Concordance of randomly repeated referral screening tools (156 of 2464) was 96% (kappa = 0.75). In comparison with the pedigree analyses, the referral screening tool demonstrated an overall (high risk by any model) sensitivity of 81.2%, specificity of 91.9%, and discriminatory accuracy of 0.87. CONCLUSIONS: Within the population studied, the referral screening tool seems to be a reliable and valid tool to identify individuals who should be referred for consideration of BRCA1/2 testing. Further examination of the referral screening tool in primary care settings is warranted to assess its impact on the efficiency with which health care providers triage patients to cancer genetics services.

Protocols for cytogenetic studies of human embryonic stem cells.

All cultured cells develop chromosome changes over time, including cultures of human embryonic stem cells (hESC), but only those cells with adaptive chromosomes changes survive. The most frequent chromosome changes in hESC cultures are trisomy 12 and trisomy 17. Cells with these trisomies are indistinguishable from normal cells by appearance and also demonstrate typical markers of pluripotency, making them difficult to identify without cytogenetic analysis. Early detection of these cells is essential since cells with trisomy 12 and 17 can replace the normal cell population in 5-10 passages. Cytogenetic analysis using G-banding is considered to be the gold standard for detecting chromosome abnormalities and, when used in combination with interphase FISH, provides a sensitive method for early detection of cytogenetic aberrations, such as full and partial trisomies of chromosomes 12 and 17. The following discussion describes the cytogenetic methods used in our laboratory to study cultured hESCs, along with recommendations for integrating these methods into a plan for routine cell line quality control.

Isolation and characterization of novel rhesus monkey embryonic stem cell lines.

ESCs are important as research subjects since the mechanisms underlying cellular differentiation, expansion, and self-renewal can be studied along with differentiated tissue development and regeneration in vitro. Furthermore, human ESCs hold promise for cell and tissue replacement approaches to treating human diseases. The rhesus monkey is a clinically relevant primate model that will likely be required to bring these clinical applications to fruition. Monkey ESCs share a number of properties with human ESCs, and their derivation and use are not affected by bioethical concerns. Here, we summarize our experience in the establishment of 18 ESC lines from rhesus monkey preimplantation embryos generated by the application of the assisted reproductive technologies. The newly derived monkey ESC lines were maintained in vitro without losing their chromosomal integrity, and they expressed markers previously reported present in human and monkey ESCs. We also describe initial efforts to compare the pluripotency of ESC lines by expression profiling, chimeric embryo formation, and in vitro-directed differentiation into endodermal, mesodermal, and ectodermal lineages.

Pre- and postnatal findings in trisomy 17 mosaicism.

Trisomy 17 mosaicism is one of the rarest autosomal trisomies in humans. Thus far, only 23 cases have been described, most of them detected prenatally. In only five instances has mosaicism been demonstrated in lymphocytes and/or fibroblasts postnatally, and only in these have multiple congenital anomalies (MCA), facial dysmorphisms, and mental retardation been reported. Patients with trisomy 17 mosaicism at amniocentesis and a normal karyotype in blood and fibroblasts (n = 17) were always healthy. Here, we report on pre- and postnatal clinical, cytogenetic, molecular-cytogenetic, and molecular findings in four patients with trisomy 17 mosaicism. The first case was detected in cultured but not in short-term chorionic villi and amniocytes. Due to MCA on prenatal ultrasound examination the pregnancy was terminated. The second patient is a 13-month-old healthy boy, in whom low level trisomy 17 mosaicism was detected in cultured chorionic villi only. The third patient is a 2-year-old girl with growth retardation, developmental delay, MCA, and trisomy 17 mosaicism in amniocytes, fibroblasts, and placenta, but not in blood and buccal smear. The fourth patient is a 9-year-old boy with growth and mental retardation, sensoneurinal hearing loss, and MCA. Cytogenetic analyses showed trisomy 17 mosaicism in amniocytes, skin fibroblasts, and urinary sediment cells, whereas in blood and buccal smear a 46,XY karyotype was found. Molecular investigations in all four cases indicated biparental inheritance of chromosome 17. Formation of trisomy was most likely due to a maternal meiosis I error in Patient 1 and a postzygotic non-disjunction of the paternal chromosome 17 in Patient 4. Cerebellar malformations, reported in two cases from the literature and in two reported here may be a specific feature of trisomy 17 mosaicism. Since the aberration has rarely been reported in lymphocytes, chordocentesis is not indicated in prenatal diagnosis. Prenatal genetic counseling for trisomy 17 mosaicism in chorionic villi or amniocytes should consider that the clinical significance remains uncertain.

Embryonic and extraembryonic stem cell lines derived from single mouse blastomeres.

The most basic objection to human embryonic stem (ES) cell research is rooted in the fact that ES cell derivation deprives embryos of any further potential to develop into a complete human being. ES cell lines are conventionally isolated from the inner cell mass of blastocysts and, in a few instances, from cleavage stage embryos. So far, there have been no reports in the literature of stem cell lines derived using an approach that does not require embryo destruction. Here we report an alternative method of establishing ES cell lines-using a technique of single-cell embryo biopsy similar to that used in pre-implantation genetic diagnosis of genetic defects-that does not interfere with the developmental potential of embryos. Five putative ES and seven trophoblast stem (TS) cell lines were produced from single blastomeres, which maintained normal karyotype and markers of pluripotency or TS cells for up to more than 50 passages. The ES cells differentiated into derivatives of all three germ layers in vitro and in teratomas, and showed germ line transmission. Single-blastomere-biopsied embryos developed to term without a reduction in their developmental capacity. The ability to generate human ES cells without the destruction of ex utero embryos would reduce or eliminate the ethical concerns of many.

Human embryonic stem cells derived without feeder cells.

BACKGROUND: Human embryonic stem cells are likely to play an important role in the future of regenerative medicine. However, exposure of existing human embryonic stem-cell lines to live animal cells and serum risks contamination with pathogens that could lead to human health risks. We aimed to derive an embryonic stem-cell line without exposure to cells or serum. METHODS: Frozen cleavage-stage embryos were thawed and cultured to the blastocyst stage. Inner cell masses were isolated by immunosurgery and plated onto extracellular-matrix-coated plates that can be easily sterilised. Six established human embryonic stem-cell lines were also maintained with this serum and feeder free culture system. FINDINGS: A new stem-cell line was derived from human embryos under completely cell and serum free conditions. The cells maintained normal karyotype and markers of pluripotency, including octamer binding protein 4 (Oct-4), stage-specific embryonic antigen (SSEA)-3, SSEA-4, tumour-rejection antigen (TRA)-1-60, TRA-1-81, and alkaline phosphatase. After more than 6 months of undifferentiated proliferation, these cells retained the potential to form derivatives of all three embryonic germ layers both in vitro and in teratomas. These properties were also successfully maintained (for more than 30 passages) with the established stem-cell lines. INTERPRETATION: This system eliminates exposure of human embryonic stem cells and their progeny to animal and human feeder layers, and thus the risk of contamination with pathogenic agents capable of transmitting diseases to patients.

Expression of the receptor tyrosine kinase Axl promotes ocular melanoma cell survival.

Metastatic tumor cells originating from cancers of a variety of tissues such as breast, skin, and prostate may remain dormant for long periods of time. In the case of uveal melanoma, the principal malignancy of the eye, complete removal of the primary tumor by enucleation can nonetheless be followed by metastatic tumor growth in distant organs months, years, or even decades later. This suggests that tumor cells have already spread to secondary sites at the time of treatment and remain dormant as micrometastases. Identifying factors that govern long-lived survival of metastatic tumor cells is therefore key to decreasing mortality associated with this and other diseases. While investigating factors differentially expressed in melanoma cells and normal melanocytes, we identified the receptor tyrosine kinase Axl and found up-regulation of Axl in uveal melanomas and melanoma cell lines by RNase protection, Western analysis, and immunohistochemistry. Axl has been shown to mediate cell growth and survival through its ligand Gas6 in non-transformed cells. To test whether stimulation of Axl can enhance survival of uveal melanoma cells, we assessed the degree of mitogenesis and cell survival by bromodeoxyuridine incorporation and trypan blue exclusion, respectively, upon stimulation of Mel 290 uveal melanoma cells with Gas6 in vitro. We show that Gas6 mediates mitogenesis and cell survival in Mel 290 cells. We further demonstrate that these effects occur specifically through the Axl receptor by modulating the expression of Axl with an antisense construct. cDNA microarray analysis of 12,687 genes then revealed that Gas6 stimulation of Axl in Mel 290 cells results primarily in the down-regulation of Cyr61, a member of the CCN protein family involved in tumor progression. These data show that the Axl pathway mediates increased survival of uveal melanoma cells, potentially advantageous during cancer dormancy, and that Axl may function in part through regulation of Cyr61.

Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells.

We have observed karyotypic changes involving the gain of chromosome 17q in three independent human embryonic stem (hES) cell lines on five independent occasions. A gain of chromosome 12 was seen occasionally. This implies that increased dosage of chromosome 17q and 12 gene(s) provides a selective advantage for the propagation of undifferentiated hES cells. These observations are instructive for the future application of hES cells in transplantation therapies in which the use of aneuploid cells could be detrimental.

Localization of transfected B7-1 (CD80) DNA in human melanoma cells after particle-mediated gene transfer.

The purpose of this study was to evaluate stable DNA transfection of M-21 human melanoma cells with particle-mediated gene transfer (PMGT) with B7-1 cDNA and to identify sites of gene integration. Stable B7-1 transfectants (M-21-B7) were obtained with PMGT using a plasmid vector containing cDNA for both B7-1 and neomycin phosphotransferase, with subsequent selection with G418. The transfected cells were flow sorted by B7-1 expression into two populations, bright and dim. The bright population had 85%-90% of cells expressing B7-1; the dim population had less than 50% of cells with B7-1 expression. Chromosome analysis with fluorescence in situ hybridization (FISH) and G-banding showed that 70% of bright cells had two main integration sites, with extensive amplification of the transgene. The dim population had random signal distribution, with little or no amplification, despite G418 selection. Because B7-1 has been mapped to 3q21, FISH was performed using a chromosome 3 painting probe (WCP) together with a probe for B7-1. In transfected bright M-21 cells, amplified genes that hybridized with the B7-1 construct were localized to chromosome 3 material inserted into marker chromosomes. These data suggest that B7-1 insertion may involve homologous recombination, but maintenance of integration and amplification required selection.

Expression of angiogenic factors Cyr61 and tissue factor in uveal melanoma.

OBJECTIVE: To study the expression of angiogenic factors Cyr61 and tissue factor (TF) in uveal melanoma and its correlation with blood vessel density. METHODS: Suppression subtractive hybridization was used to identify genes that are differentially expressed between cell lines of uveal melanoma and normal uveal melanocytes. Expression of these genes was subsequently verified in primary uveal melanomas and correlated with the number of blood vessels in archival specimens by immunohistochemical analysis. RESULTS: Cyr61 and TF are expressed at elevated levels in cell lines of uveal melanoma compared with normal uveal melanocytes. Duplication of a region of chromosome arm 1p, encompassing the genes encoding Cyr61 and TF, was observed in the melanoma cell line used in the initial subtractive hybridization. Both genes are also expressed in primary uveal melanomas, and a correlation was found between expression of TF and the number of blood vessels in archival specimens. CONCLUSIONS: Cyr61 and TF may contribute to the angiogenic phenotype associated with uveal melanoma. A region of chromosome arm 1p also may contain oncogenes or tumor suppressor genes pertinent to the origins of this type of ocular tumor. CLINICAL RELEVANCE: New immunotherapies have been devised for the treatment of cancer based on the expression of TF. Similar approaches may be effective in treating uveal melanoma.