Current Human Embryonic Stem Cell Lines Contaminated
With Potentially Dangerous Non-Human Molecule, UCSD/Salk Team
By Sue Pondrom
lines of human embryonic stem cells have been contaminated with
a non-human molecule that compromises their potential therapeutic
use in human subjects, according to research by investigators
at the University of California, San Diego (UCSD) School of
Medicine and the Salk Institute in La Jolla, California.
In a study published
online January 23, 2005 in the journal Nature Medicine,
the researchers found that human embryonic stem cells, including
those currently approved for study under federal funding in
the U.S., contain a non-human, cell-surface sialic acid called
N-glycolylneuraminic acid (Neu5Gc), even though human cells
are genetically unable to make it. In a related paper published
November 29, 2004 by the Journal of Biological Chemistry
(JBC), the Varki group has also discovered the exact cellular
mechanism by which this occurs.
In studies with one
of the federally approved human embryonic stem cell lines, the
investigators determined that the Neu5Gc is incorporated by
the stem cells when they are grown or derived from laboratory
cultures that contain animal sources of the non-human Neu5Gc
molecule. All traditional culture-dish methods used to grow
all human embryonic stem cells include animal-derived materials,
including connective tissue cells (so-called “feeder layers”)
from mice and fetal calf serum.
UCSD professor of Medicine, and cellular & molecular
“The human embryonic
stem cells remained contaminated by Neu5Gc even when grown in
special culture conditions with commercially available serum
replacements, apparently because these are also derived from
animal products,” said both papers’ senior author
Ajit Varki, M.D., UCSD professor of medicine and cellular &
molecular medicine, and co-director of the UCSD Glycobiology
Research and Training Center.
The research in Nature
Medicine was done with human embryonic stem cells grown
in the laboratory of Fred Gage, Ph.D., professor, Laboratory
of Genetics, the Salk Institute, La Jolla, California, an adjunct
professor of neurosciences at UCSD, and an author on the Nature
Previously, the Varki
lab found in 1998 that humans are uniquely different from other
mammals studied in that people do not express Neu5Gc*.
In a 2003 study**, the UCSD researchers found
that humans have naturally occurring antibodies that are directed
against Neu5Gc. In the current Nature Medicine paper,
the scientists found that the human embryonic stem cells contaminated
with Neu5Gc became, effectively, like animal cells, being attacked
by human antibodies, and thus rendering them useless as a potential
therapeutic tool in humans.
"It's an important
safety issue because this opens up the idea that metabolic transfer
of glycans is occurring between cells," said Gage. "Also,
components of the growth medium have the capacity to change
the immunological characteristics of the human ES cells. More
research is needed to understand the optimal conditions for
preparing human cells for therapeutic application."
that one partial solution to the problem was to use human serum
in the growth medium,” Varki said. When the team grew
the cells in heat-inactivated human serum specially selected
for low concentrations of anti-Neu5Gc antibodies, the immune
response was significantly reduced, but not completely eliminated.
In their experiments,
the researchers used recently developed probes to detect the
presence of Neu5Gc on the cell surface of human embryonic stem
cells that had been grown in traditional culture conditions.
The scientists further confirmed the presence of Neu5Gc with
a process called electrospray mass spectrometry. The percentage
of total sialic acids present as Neu5Gc in the embryonic stem
cells varied from 2.5 to 10.5 percent. In human embryonic stem
cells that had been allowed to differentiate into embryoid bodies
(EB), which is the first step in preparing them for potential
use in humans, the percentage of total sialic acids present
still ranged from 5 to 17 percent.
Varki and his team
noted that many efforts have been made during the last few years
to try to eliminate any animal-derived culture components in
human stem cell culture. However, many of the specialized media
used for growth and differentiation still contain materials
from animal sources and are hence contaminated with Neu5Gc.
In addition to using
human serum, the researchers suggested the possibility of using
what are called “feeder cells” from mice with a
human-like defect in Neu5Gc production. They noted that they
have recently produced such a mouse. Another possibility being
attempted by groups in other parts of the world is to use human
embryo-derived connective tissue cells as the feeder layer in
A further solution
might be a short-term culture in heat-inactivated serum from
the actual patient who is going to receive the therapy, the
scientists said. However, it may still prove difficult to completely
eliminate the Neu5Gc, because is has become metabolically incorporated
into the currently available, federally-funded human embryonic
stem cell lines.
“With this discovery,
the preexisting general concern about using animal products
for deriving human embryonic stem cells has become more specific,
being defined in molecular terms,” Varki said.
will, of course, become irrelevant if complete elimination of
Neu5Gc can be achieved by deriving new human embryonic stem
cells that have never been exposed to Neu5Gc-containing animal
products of any kind,” the researchers said in the Nature
Medicine paper, noting that none of the suggested approaches
guarantees the complete elimination of Neu5Gc from existing
cultures. “Therefore, it would seem best to start over
again with newly derived human embryonic stem cells that have
never been exposed to any animal products, and ideally, only
ever exposed to serum from the intended transplant recipient.”
an approach could not be pursued under existing rules for the
use of federal grant dollars,” Varki said.
The first author of
the Nature Medicine study is Maria J. Martin, Ph.D.,
a post doctoral researcher in Varki’s lab at UCSD. An
important additional author is Gage’s post doctoral fellow
Alysson Muotri, Ph.D. The study was funded by the National Institute
of General Medical Sciences at the National Institutes of Health,
the Lookout Fund, and by the G. Harold and Leila Y. Mathers
Charitable Foundation of New York.
In addition to Varki,
authors of the related study in JBC included Muriel
Bardor, Ph.D. and Dzung Nguyen, Ph.D., post-doctoral fellows,
and Sandra Diaz, a research associate. They determined that
Neu5Gc gets into human cells by being engulfed in fluid droplets
and then moved to the cytoplasm of the cell by a “pump”
called the lysosomal sialic acid transporter.
Varki noted that this
pathway is an unusual and previously unknown one that may also
be relevant to the entry of other small molecules into cells.
In addition, the JBC study showed how Neu5Gc attached
to dietary proteins from animals could be incorporated into
cells lining the stomach and colon, organs where consumption
of red meat has been associated with risk of cancer.
mechanism that this molecule uses to get into human cells may
give us clues to possible solutions to the problems that it
may cause in various situations,” Varki said.
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Media Contact: Sue Pondrom