Embryonic Stem Cell Lines Accumulate Potentially Dangerous Mutations

Existing embryonic stem cell lines that have divided for years contain many genetic mutations that earlier versions of those lines do not contain.

An international team of researchers has discovered that human embryonic stem cell lines accumulate changes in their genetic material over time.

The findings do not limit the utility of the cells for some types of research or for some future clinical applications, the researchers say, but draw attention to the need to closely monitor stem cell lines for genetic changes and to study how these alterations affect the cells' behavior. The researchers' work is described in the Sept. 4 online edition of Nature Genetics.

"This is just the first step," says Aravinda Chakravarti, Ph.D., one of the research team's leaders and professor and director of the McKusick-Nathans Institute of Genetic Medicine at Johns Hopkins. "While this is a snapshot of the genomic changes that can happen, it's certainly not everything going on. We still need comprehensive analyses of the changes and what they mean for the functions of embryonic stem cells."

"Embryonic stem cells are actually far more genetically stable than other stem cells, but our work shows that even they can accumulate potentially deleterious changes over time," adds Anirban Maitra, M.B.B.S., an assistant professor of pathology at Johns Hopkins who shares first authorship of the paper with Dan Arking, Ph.D., an instructor at Hopkins. Both are members of the McKusick-Nathans Institute of Genetic Medicine at Johns Hopkins. "Now it will be important to figure out why these changes occur, how they affect the cells' behavior and how time affects other human embryonic stem cell lines."

My guess is they compared versions of the stem cell lines that had been frozen years ago with other versions of those same lines that had been kept growing in cell culture dividing many times since each embryonic stem cell line was created. Those sort of sub-lines of the original stem cell lines that have divided more have more mutations. Note that this is not surprising. Cells grown in culture are not growing in ideal conditions and when cells divide they do so imperfectly anyhow.

The researchers in the United States, Singapore, Canada and Sweden compared "early" and "late" batches of each of nine federally approved human embryonic stem cell lines. Twenty-nine human embryonic stem cell lines from seven different companies are approved by the United States National Institutes of Health under President George W. Bush's policy restricting federal funding of this research to cell lines in existence before his announcement of the policy at 9 p.m. ET, Aug. 9, 2001. The dozens of human embryonic stem cell lines developed since that announcement cannot be used in federally funded research.

Most of the "late" batches of stem cells -- those grown in the lab a year to three years longer than their early counterparts -- displayed gross changes in the number of copies of chromosomes or parts of chromosomes, in the marks that control whether a gene is used by the cell, or in the sequence of DNA found in the cell's mitochondria.

Some of the changes found resemble changes seen in cancer cells.

"The majority of the lines we tested had genetic changes over time," says Chakravarti. "Whenever you have something in a culture dish, it can change, and it will be important to identify, keep track of and understand these changes."

At this point, the precise effects of these changes on the cells aren't known, but some of the changes resemble those seen in cancerous cells. At any rate, the changes presumably became entrenched in a particular cell line because they conferred some advantage as the cells were grown in laboratory dishes. Whether the changes affect the stem cells' abilities to become other cell types is also unknown.

In the body aged adult stem cells that accumulate dangerous mutations are suspected by many scientists as being major sources of cancer. Adult stem cells grown in culture will mutate just as these embryonic stem cells have done. Therefore this result does not demonstrate a problem specific to embryonic stem cells and should not be seen as a useful debate point by opponents of human embryonic stem cell research.

Note how the scientists can not say for sure whether any of the mutations in these human embryonic stem cells put them at risk of causing cancers. One reason for this lack of certainty is that all the genetic mutations that contribute to cancer are not yet known. The other reason is that even if all those mutations were known some might be hard to test for. To defeat cancer and fully realize the potential of both adult and embryonic stem cells we need cheaper and better technologies for DNA sequencing and DNA testing.

Gene chips were essential tools for this research. Better tools mean better and faster research.

Although research with human embryonic stem cells is still in the lab -- not the clinic -- focusing on what the cells can do and how they are controlled, the hope is that in the future these cells might help replace or repair tissues lost to disease or injury. Because embryonic stem cells can become any type of cell found in the body, in theory they could replace certain pancreas cells in people with type I diabetes, or regenerate brain cells lost in a person with Parkinson's disease, for example.

The analyses of the embryonic stem cell lines and the computer comparisons of the mounds of resulting data required the efforts of scientists at four academic centers, two federal laboratories and three companies. Critical to the team's success was prescient support of cutting-edge technology development by the National Institutes of Health, support that enabled development of the technological infrastructure necessary for large-scale comparative research, particularly the Human Genome Project, says study co-author Mahendra Rao, M.B.B.S., Ph.D., of the Laboratory of Neurosciences at the National Institute on Aging.

The scientists used so-called GeneChip microarrays, or oligonucleotide arrays, to determine whether there were genetic differences between the early and the late batch of each of the stem cell lines, including whether any genes were present in extra copies. Depending on the gene affected, extra copies could lead to accelerated cell growth, increased cell death, or no measurable effect at all.

Epigenetic changes in the form of methylation patterns on the DNA backbone were also seen along with the genetic mutations. Note that epigenetic changes are also thought to contribute to the development of cancer.

In addition to probing changes in the nuclear and mitochondrial DNA sequences and copy numbers, the researchers examined whether the cells' genetic material had shifts in marks that sit on genes and are passed from cell to cell during cell division. These so-called epigenetic marks -- in this case methyl groups on a gene region known as the promoter -- help control whether a gene is used by a cell to make proteins. The researchers determined the methylation status of 14 genes in each of the batches of stem cells; three of the genes did show different methylation patterns in late batches compared to early batches.

What creates the differences between embryonic stem cells, adult stem cells, and various specialized functional cell types throughout the body? Epigenetic changes. If we had the ability to precisely change methylation patterns in any way desired then we probably could convert any cell type to any other cell type. The point is that epigenetic state is important and the development of better abilities to test and change epigenetic state would greatly help in the development of stem cell therapies.

The embryonic stem cells also had deletion and duplication mutations.

The scientists' analysis revealed that five of the nine cell lines had extra or fewer copies of at least one section of their genetic material in the late batch compared to the same cell line's early batch. Two of the nine lines had changes in their mitochondrial DNA over time, and all nine stem cell lines exhibited some shift in methylation of at least one of three genes. One of these genes, called RASSF1A, is also methylated in many cancers, but what effect the methylation has on the stem cells is unknown.

The team is already planning to conduct similar analyses of the remaining NIH-approved cell lines, but analysis of stem cell lines not available for use with federal funds will also be needed, the team members say.

These results suggest that existing embryonic stem cell lines are going to have limited utility in the development of therapies. Lots of research can still be conducted on these stem cell lines. But I'd be very reluctant to have any of these mutated embryonic stem cells injected into yours truly. Also, years will go by before these stem cells can get massaged into useful forms for therapies and they will accumulate even more mutations in that time.

Stem cell lines created just when they are needed (whether embryonic or slightly more differentiated adult stem cell lines) would reduce the risk of mutations. However, even "just in time" stem cell lines would need extensive genetic testing because whichever cell would be used for the starter nucleus might contain mutations that put the resulting stem cell line at heightened risk of creating a cancer.

It is possible that future gene therapies will allow at least partial repair of these cell lines. But those gene therapies could be many years into the future.

Biogerontologist Aubrey de Grey's proposal for dealing with the cancer risk from stem cell lines is to knock out the telomerase gene so that any cancer would eventually be halted by telomere decay. The downside of such an approach is that the youthful stem cell line would not function for as long in the body before needing yet another replenishment by another youthful stem cell line. But maybe that would be worth the lowered cancer risk.

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