Cultured human embryonic stem cell (hESC) lines are an invaluable resource because they provide a uniform and stable genetic system for functional analyses and therapeutic applications. Nevertheless, these dividing cells, like other cells, probably undergo spontaneous mutation at a rate of 10(-9) per nucleotide. Because each mutant has only a few progeny, the overall biological properties of the cell culture are not altered unless a mutation provides a survival or growth advantage. Clonal evolution that leads to emergence of a dominant mutant genotype may potentially affect cellular phenotype as well. We assessed the genomic fidelity of paired early- and late-passage hESC lines in the course of tissue culture. Relative to early-passage lines, eight of nine late-passage hESC lines had one or more genomic alterations commonly observed in human cancers, including aberrations in copy number (45%), mitochondrial DNA sequence (22%) and gene promoter methylation (90%), although the latter was essentially restricted to 2 of 14 promoters examined. The observation that hESC lines maintained in vitro develop genetic and epigenetic alterations implies that periodic monitoring of these lines will be required before they are used in in vivo applications and that some late-passage hESC lines may be unusable for therapeutic purposes.

Down syndrome (DS) is caused by the presence of an extra copy of human chromosome 21 (Hsa21) and is the most common genetic cause for developmental cognitive disability. The regions on Hsa21 are syntenically conserved with three regions located on mouse chromosome 10 (Mmu10), Mmu16 and Mmu17. In this report, we describe a new mouse model for DS that carries duplications spanning the entire Hsa21 syntenic regions on all three mouse chromosomes. This mouse mutant exhibits DS-related neurological defects, including impaired cognitive behaviors, reduced hippocampal long-term potentiation and hydrocephalus. These results suggest that when all the mouse orthologs of the Hsa21 genes are triplicated, an abnormal cognitively relevant phenotype is the final outcome of the elevated expressions of these orthologs as well as all the possible functional interactions among themselves and/or with other mouse genes. Because of its desirable genotype and phenotype, this mutant may have the potential to serve as one of the reference models for further understanding the developmental cognitive disability associated with DS and may also be used for developing novel therapeutic interventions for this clinical manifestation of the disorder.

DNA methyltransferase 1 (DNMT1)-deficient mice are tumorprone, and this has been proposed to result from the induction of genomic instability. To address whether loss of DNMT1, or the related protein DNMT3b, results in genomic instability in human cancer cells, we used a near-diploid human colorectal cancer cell line, HCT116, in which one or both DNMT genes were disrupted by homologous recombination. Array-based comparative genomic hybridization analyses indicated that double, but not single, DNMT knock-out cells display two specific alterations in regional DNA copy number, suggesting that DNMT deficiency and genomic DNA hypomethylation are not associated with widespread genomic amplifications or deletions in human cancer cells. However, spectral karyotype analyses revealed that DNMT-deficient HCT116 cells are highly unstable with respect to large-scale chromosomal alterations; furthermore, this effect is characterized by a high degree of individual cell heterogeneity. The induction of chromosomal alterations in DNMT-deficient cells was evidenced both by aneuploidy and by large increases in the number of novel chromosomal translocations. Studies of double knock-out cells indicated that the generation of chromosomal alterations is spontaneous and persistent in vitro, meeting the formal definition of genomic instability. In summary, we show that DNMT deficiency in human cancer cells results in constitutive genomic instability manifested by chromosomal translocations. (Cancer Res 2005; 65(19): 8635-9)