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, 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," added Anirban Maitra, an assistant
professor of pathology at Johns Hopkins who shares first
authorship of the paper with Dan Arking, an instructor at
Hopkins. Both are members of the McKusick-Nathans Institute
of Genetic Medicine. "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."
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 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.
"The majority of the lines we tested had genetic
changes over time," Chakravarti said. "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.
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 1 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, said study co-author Mahendra Rao, 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.
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.
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
said.
The Johns Hopkins researchers were funded by the Henry
J. Knott Professorship in Genetic Medicine, Sol Goldman
Pancreatic Cancer Research Center at Johns Hopkins,
National Cancer Institute, Maryland Cigarette Restitution
Fund and Donald W. Reynolds Foundation Clinical
Cardiovascular Research Center at Johns Hopkins.
Authors on the paper from Johns Hopkins are Maitra,
Arking, Morna Ikeda, Keyaunoosh Kassauei, Guoping Sui,
David Cutler and Chakravarti.
The microarrays used in this work are the product of
Affymetrix, Santa Clara, Calif. Chakravarti is a paid
member of the Affymetrix scientific advisory board. The
terms of this arrangement are being managed by The Johns
Hopkins University in accordance with its
conflict-of-interest policies.