Lorraine F. Meisner

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.

Microdeletions in the Y Chromosome of Infertile Men

Background Some infertile men with azoospermia or severe oligospermia have small deletions in regions of the Y chromosome. However, the frequency of such microdeletions among men with infertility in general is unknown. We sought to determine the prevalence of Y-chromosome microdeletions among infertile men and to correlate the clinical presentation of the men with specific deletions. Methods We studied 200 consecutive infertile men. Each man was evaluated comprehensively for known causes of infertility, and Y-chromosome microdeletions were studied with use of the polymerase chain reaction to amplify specific regions of the chromosome. The Y chromosomes of 200 normal men were also analyzed. Results Fourteen infertile men (7 percent) and four normal men (2 percent) had microdeletions of the Y chromosome. Nine of the infertile men had azoospermia or severe oligospermia (sperm concentration, <5 million per milliliter), four had oligospermia (sperm concentration, 5 million to <20 million per milliliter), and one had normospermia (sperm concentration, ≥20 million per milliliter). The size and location of the deletions varied and did not correlate with the severity of spermatogenic failure. The fathers of six infertile men with microdeletions were studied; two had the same deletions as their sons, and four had no deletions. Conclusions A small proportion of men with infertility have Y-chromosome microdeletions, but the size and position of the deletions correlate poorly with the severity of spermatogenic failure, and a deletion does not preclude the presence of viable sperm and possible conception.

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.

Preserving the genetic integrity of human embryonic stem cells

transactions are the difficulty in accessing correct and complete information on potential partners, suppliers or market possibilities and the uncertainty of ensuring a partner’s commitment to formal contracts. The latter in particular, requires a judicial system that functions more efficiently and credibly. These conditions have nothing to do with TRIPS. Thus, the impact of TRIPS on either the commercial strategies of foreign companies or their strategic alliances with Indian companies is anyone’s guess, as it is only one parameter among many that will be used in making foreign investment decisions. From the perspective of Western firms, the implementation of TRIPS in India may encourage them to introduce new brand drugs because such products will now enjoy patent protection—a situation not possible since 1970. This will not mean, however, that high-priced, Western-manufactured products can be directly shoehorned into the Indian market. As K.S.N. Prasad, CEO of Shantha Biotechnics (Hyderabad, India), puts it: “Though TRIPS gives exclusive rights to Western companies to market their brand products in India—eliminating competition from local companies that copy inventions— these multinationals are unlikely to benefit from selling their products at high prices because Indian consumers simply cannot afford the high costs of drugs developed and manufactured abroad. Therefore, it will be necessary for Western and Indian companies to enter into strategic alliances so that novel drugs can be manufactured under license for local consumption. Such alliances will lead to a win-win situation for all, both biotech companies and the public.” To sum up, Indian biotech firms basically have three choices in the short-term as business innovation strategies2: first, they can focus on products that are either off-patent already or soon to be off-patent (essentially the generics market); second, they can collaborate with Western multinationals and biotech companies (two areas that are likely to witness an increase in collaborations are clinical trials and R&D outsourcing); or third, they can focus on innovations that the multinationals will not be interested in; that is, mainly ‘tropical’ or developing world diseases.

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.

Cyanobacteria (blue-green algae) in Wisconsin waters: acute and chronic toxicity.

Abstract Toxins produced by several species of cyanobacteria (blue-green algae) are a potentially serious environmental problem. These substances can be acutely toxic as evidenced by the death of livestock and other animals that have been exposed to them. In this study, samples collected from 102 sites in Wisconsin were analyzed for toxicity. Acute toxicity was tested by intraperitoneally injecting mice with lysed algal cells (identified to genus) and observing the effects. Chronic toxicity was tested using the Salmonella typhimurium mutagenicity test, the Bacillus subtilis multigene sporulation test and a chromosome breakage test using human lymphocytes. Mouse bioassay results showed that about 25% of the sites contained toxic algae, indicating acutely toxic algae are commonplace in Wisconsin waters. Bacterial assay results suggested the toxins were not directly mutagenic, but a chromosomal breakage test suggested the possibility that the algal toxins may be clastogenic. Overall, the results indicate that algal toxins may be more serious environmental hazards than generally recognized.

A Juvenile Polyposis Tumor Suppressor Locus at 10q22 Is Deleted From Nonepithelial Cells in the Lamina Propria

BACKGROUND & AIMS Juvenile polyps are characterized by an abundant lamina propria that lacks smooth muscle and may contain cystically dilated glands, with epithelium that seems normal and is nondysplastic. Rarely, an autosomal dominant inheritance pattern occurs. The aim of this study was to test the hypothesis that the genetic defect in both sporadic juvenile polyps and hereditary juvenile polyposis involves loss of function for a tumor suppressor gene. METHODS Allelic losses were detected by comparing normal DNA with tumor DNA from a series of 47 juvenile polyps from 16 patients using polymerase chain reaction amplification of microsatellite markers and fluorescent in situ hybridization (FISH). RESULTS Somatic deletions at 10q22 were detected in 39 of 47 juvenile polyps (83%) from 16 unrelated patients with either hereditary or sporadic juvenile polyps, and the minimum overlap localized juvenile polyposis coli to the 3-cM interval D10S219-D10S1696. Fluorescent in situ hybridization shows that the cells affected by deletion mutation reside exclusively in the lamina propria, not in the epithelium. CONCLUSIONS The location of a novel tumor suppressor gene on chromosome 10 that is affected by deletion mutation in the majority of juvenile polyps was mapped. Unlike adenomas and carcinomas of the colonic epithelium, juvenile polyps originate in the lamina propria.

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.

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.

Synovial sarcoma of the upper digestive tract: a report of two cases with demonstration of the X;18 translocation by fluorescence in situ hybridization.

Two cases of synovial sarcoma that arose in the upper digestive tract are reported. One case was a polypoid mass that arose at the gastroesophageal junction; the other was a large intramural mass that arose in the wall of the stomach. Both cases had a classic biphasic pattern. In the stomach tumor, the biphasic morphology was focal and there was an abrupt transition to poorly differentiated synovial sarcoma. The tumors had immunohistochemical features that were consistent with synovial sarcoma. Ultrastructural evaluation of the gastroesophageal tumor supported the diagnosis. The diagnostic X;18 translocation was demonstrated by fluorescence in situ hybridization on sections from paraffin-embedded tissue in 86% and 50% of interphase nuclei from the gastroesophageal and gastric tumor, respectively. The translocation was present in equal frequency in the epithelial and spindle cells in the biphasic areas and the poorly differentiated areas of the gastric tumor, indicating that the development of the more aggressive subclone was probably due to genetic mutations not encompassing the SYT-SSX gene fusion product. We are aware of only five reported cases of synovial sarcoma arising in the digestive tract, all in the proximal esophagus. These cases are the first reported arising in the gastroesophageal junction and stomach and the only cases of synovial sarcoma of the digestive tract in which the diagnostic translocation was demonstrated. Sarcomatoid carcinoma (carcinosarcoma) and gastrointestinal stromal tumor are the main differential diagnoses for synovial sarcoma in this site. Synovial sarcoma of the digestive tract may be underdiagnosed, and its recognition may have important clinical implications. Fluorescence in situ hybridization is helpful in making this distinction.