Embryonic stem lines unstable

Genomic changes could render federally appointed lines unusable therapeutically, says Nature Genetics report

By | September 6, 2005

Human embryonic stem cells accrue changes in their genomes that could make them unusable therapeutically when cultured at length, an international team of scientists report in the October Nature Genetics.

"Some of the early embryonic stem cell passages were relatively aberration-free, at least using the technology we have access to, even at passage 30 or so, which would be unusual for most adult stem cells," coauthor Anirban Maitra of Johns Hopkins University School of Medicine in Baltimore told The Scientist. Still, "over time, it appears that the majority of even embryonic stem cells do accumulate genetic and epigenetic changes."

Coauthor Mahendra Rao, head of the stem cell group at the National Institute on Aging's neuroscience lab in Baltimore, and colleagues had previously found neural stem cells became karyotypically abnormal in culture, corroborating widespread reports that adult stem cells accumulate genetic aberrations. "They lose their stemness," Rao told The Scientist.

In contrast, embryonic stem cells were thought to be far more genetically stable than adult stem cells, Maitra said. He and colleagues in Sweden, Canada, Singapore, and the United States analyzed early- and late-passage cultures of nine human embryonic stem cell lines approved for use by the US federal government. The late batches were cultured 22 to 175 passages more than early counterparts, or roughly a year to three years longer.

Oligonucleotide arrays containing roughly 115,000 single nucleotide polymorphisms revealed that four late-passage lines developed copy number aberrations. These ranged from large genomic amplifications or deletions, such as amplification of the entire 17q arm, to more discrete changes, such as a two-megabase amplification encompassing the MYC oncogene.

The researchers found mitochondrial DNA sequence alterations in two late-passage lines, with five changes occurring in the coding region. Three of these resulted in missense mutations in mitochondrially encoded NADH dehydrogenases 1, 2 and 4, and one caused a nonsense mutation in ATPase 6. Real-time quantitative methylation-specific PCR assays of 14 genes with known differential methylation patterns in cancer tissues, such as putative tumor suppressor RASSF1A, revealed higher promoter methylation levels in eight late-passage lines.

"The very act of culturing and repeatedly passing cells introduces a selection process, and cells that accumulate any kind of DNA modification, genetic or epigenetic, that confers an advantage for doubling are ultimately going to take over," James Battey, chair of the National Institutes of Health stem cell task force, who did not participate in this study, told The Scientist. "These new findings are very fine scientific work, and shows we need to be vigilant with these cells, especially when passaged for long periods of time."

While the researchers say late-passage lines may be unusable therapeutically, they note early-passage lines could prove useful. Future experiments should examine what effects these changes actually have phenotypically, "such as whether they make them more cancer-prone, or change their ability to differentiate," coauthor Aravinda Chakravarti, director of the McKusick-Nathans Institute of Genetic Medicine at Johns Hopkins, told The Scientist.

The mechanisms that apparently make embryonic stem cells more stable in early passages than adult ones remain uncertain and merit further study, Battey said. "Maybe they have more robust mechanisms for DNA repair," he speculated. "Certainly if a cell has a capacity to self-renew many times and differentiate into many cell types, one would imagine maintaining one's genetic fidelity is pretty important."

"The obvious issue that we and others, no doubt including the present authors, will wish to establish is what are the selective advantages of the mutations that are seen," Peter Andrews of the University of Sheffield, who did not participate in this study, told The Scientist. "Do they indeed appear in most of the cells because of selection, rather than because of, say, a population bottleneck resulting, say, from low recovery after freezing and thawing? What are the conditions that favor the appearance of changes in specific genes? What role do these genes play in the biology of the human embryonic stem cell? For instance, do they plan a role in controlling proliferation or commitment to differentiation?" Andrews continued. "These issues will both provide insights into the mechanisms that control proliferation and differentiation in human embryonic stem cells and help us design ways to minimize selection for such changes."

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