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.
"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 neuroscien
ces 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."
"We considered 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 not
ed 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 the culture.
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
"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.
"Such issues 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."
"However, such an approach could not be pursued under existing rules
for the use of federal grant dollars," Varki said.
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
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.
"Knowing the 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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