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.

FISH was an alternative technique for determining gene amplification and had some distinct advantages over Southern hybridization. Our results demonstrate that HER-2/neu gene amplification in the absence of adjuvant therapy is an independent predictor of poor clinical outcome and is a stronger discriminant than tumor size. Women with small tumors that had gene amplification were at increased risk of recurrence and disease-related death.

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We report the isolation and characterization of a spontaneously immortalized human keratinocyte cell line, NIKS. The cell line is not tumorigenic in athymic nude mice and maintains cell-type-specific requirements for growth in vitro. NIKS cells express steady-state levels of transforming growth factor-alpha, transforming growth factor-beta1, epidermal growth factor receptor, c-myc, and keratin 14 mRNAs comparable with the parental BC-1-Ep keratinocyte strain. BC-1-Ep and NIKS keratinocytes produce similar levels of cornified envelopes and nucleosomal fragmentation in response to loss of substrata attachment. DNA fingerprinting results confirm that the NIKS cells originated from the parental BC-1-Ep keratinocytes. NIKS cells contain 47 chromosomes due to an extra isochromosome of the long arm of chromosome 8, and the near-diploid karyotype appears to be stable with repeated passage. A fully stratified squamous epithelium is formed by the NIKS keratinocytes in organotypic culture. Ultrastructural analysis of both the parental and immortalized keratinocytes reveals abundant desmosomes, hemidesmosomes, and the production of a basal lamina. Our findings with the NIKS cells support the observation that spontaneous immortalization is not linked to alterations in squamous differentiation or the ability to undergo apoptosis. The NIKS human keratinocyte cell line is an important new tool for the study of growth and differentiation in stratified squamous epithelia.

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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.

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.