Editor's Note: This is part one of a two-part series on stem cell research. Part two will be published in the Feb. 1 issue.
Embryonic stem cells possibly are the most politicized subject in biomedical research. Besides the "origin of life" questions they pose, they have been hailed as a future cure for just about everything, although that belief, while fairly widespread in the scientific community, awaits clinical confirmation.
That awaited confirmation partly is due to political restrictions on federal funding for embryonic stem cell research. Federal funding is available only for research on human embryonic stem cell lines that meet certain guidelines on their origin and ones that were created prior to August 2001. The NIH said in the third quarter of 2004 that there were 22 human embryonic stem cell lines that federally supported researchers could purchase.
But the complexity of the underlying science also is daunting. Embryonic stem cells can develop into any tissue type, but getting them to predictably develop into one specific tissue type vs. another is a difficult undertaking, with the net effect that none of the approved stem cell lines has yet made it into clinical trials.
Research in the February 2005 issue of Nature Medicine, now available online, has the potential to further delay the clinical debut of NIH-approved human embryonic stem cell lines. In "Human embryonic stem cells express an immunogenic nonhuman sialic acid," researchers from the University of California at San Diego and the Salk Institute, also located in San Diego, showed that the culturing methods used to grow most embryonic stem cell lines have led to a contamination of those lines with sialic acids, a family of acidic sugars that are not produced by humans. The researchers focused their attention mainly on one specific sialic acid, known as Neu5Gc. Humans routinely eat Neu5Gc, which is abundant in milk and red meat.
It also is immunogenic, meaning that when it is eaten, it often stimulates the production of antibodies.
"Since many humans have circulating antibodies against Neu5Gc, exposure of such cells to human blood serum marks the [embryonic stem] cells for destruction," said senior author Ajit Varki, professor of medicine and cellular and molecular medicine at the University of California at San Diego. "Thus, we predict that if human embryonic stem cells or their derivatives grown under the current conditions were put into a living human, there is a significant chance of a deleterious immune reaction and/or rejection of the transplanted cells."
Embryonic stem cells routinely are co-cultured with animal cells, so-called mouse feeder layers, as well as in media containing fetal calf serum, to provide nutrients. Several commercial serum replacements are available to avoid the use of animal serum, but at least some of those also contain animal products.
When stem cells are separated from the feeder cell layers, they differentiate into cell aggregates called embryoid bodies, which still can turn into many cell types. The scientists tested both stem cells and embryoid bodies, grown under standard conditions, for the presence of sialic acids by antibody staining, and confirmed their finding with mass spectrometry. Both stem cells and embryoid bodies had incorporated sialic acids, including Neu5Gc.
The scientists then investigated the sources of Neu5Gc in the stem cells and embryoid bodies. The mouse feeder layer apparently contributed some but not all of the Neu5Gc, since separating the stem cells from the mouse feeder layer to make embryoid bodies did not eliminate the Neu5Gc. Varki and his colleagues found that in the medium used to feed the embryoid bodies, serum replacement was a major source of Neu5Gc, while medium without serum replacement contained very little Neu5Gc.
An Additional Barrier To Transplantation
Next, the team investigated whether human antibodies would recognize Neu5Gc on embryonic stem cells as foreign, which they did. Asked to compare possible problems arising from the contamination to the immunological issues arising from transplantation of the cells themselves - which are, after all, similar to an organ transplant - Varki told BioWorld Today that it isn't "possible to know at this time how severe a problem it will be," adding that "the highly variable levels of natural anti-Neu5Gc antibodies" might make for "individual variability in the severity of the reaction."
He also said, "Regardless, unlike the case with other known transplantation barriers that are polymorphic [such as blood type or HLA type] one cannot do any selective matching of specific embryonic stem cells to individual humans. Also, while somatic nuclear-cell transfer could one day solve most of the other incompatibility problems, avoiding Neu5Gc requires getting away from exposure to animal products and cells."
Once Neu5Gc has been incorporated into the stem cells, you can get the cell out of the contamination but it apparently is more difficult to get the contamination out of the cell. When the researchers tried to clean the cells by growing them in human serum specially selected for low concentrations of anti-Neu5Gc antibodies, the immune response was reduced but not eliminated, showing that the cells still contained Neu5Gc.
The authors suggested a few other possibilities for reducing the amount of Neu5Gc in contaminated stem cells, but noted that in the end, "none of these approaches guarantees the complete elimination of Neu5Gc (or any other unknown animal antigen or pathogen) from existing cultures. Therefore it would seem safest to start over again with newly derived [human embryonic stem cells] that have never been exposed to any animal products containing Neu5Gc."
They added, "the current regulatory climate in the United States precludes this type of approach, when using federal grant dollars."
A Pandemic Or Just A Local Outbreak?
It is hard to guage just how widespread the contamination is at this point. The scientists directly tested only one of the 22 embryonic stem cell lines approved for federally funded research. But in a paper published in the Nov. 22, 2004, issue of the Journal of Biological Chemistry, Varki and his colleagues described the mechanism by which Neu5Gc is incorporated into human cells. Varki noted that the mechanism "is universal to all human cell types we have studied. Thus, it is almost certain that all of the other embryonic stem cells grown in this manner would have the same contamination."
Reaction to the paper was widespread, with everybody from the NIH to biotechnology companies working in the stem cell field weighing in. Several companies, including Aastrom Biosciences Inc., ITL Laboratories LLC, MultiCell Technologies Inc. and StemCells Inc., issued statements defending their research. By and large, the companies pointed out that they use neither embryonic stem cells nor mouse feeder cells. But the non-use of mouse feeder layers does not in itself guarantee that cells are free from contamination, since at least one commercially available serum replacement tested by Varki's group contained Neu5Gc.
MultiCell Technologies said that "none of the source of the contamination, sialic acid, Neu5Gc, is present on [MultiCell] human cell lines." ITL's parent company, Epipharma Inc., told BioWorld Today that "the culture media used in our research will be made in our laboratory and will not contain animal products of non-human origin," while StemCells CEO Martin McGlynn noted that "StemCells' manufacturing process does not involve the use of animal serum, animal-derived proteins or proprietary serum replacements of unknown composition."
Varki's group also cited work from research groups outside of the U.S., including NIH-approved Swedish stem cell lines, which did not use mouse feeder layers. Those might be a source of uncontaminated stem cells, but Varki noted that source needs to be tested because of the possibility that the sera or serum replacements used to grow those cells contained animal products.
Asked whether it was possible to genetically engineer mice so that their feeder cell layers would not contain Neu5Gc, Varki said, "We have, in fact, made such cells. However, this may not be the only problem arising from exposing human embryonic stem cells to mouse materials. Thus, we prefer not to pursue this possible short-term fix, but recommend that methods be found to eliminate/replace the mouse feeder layers altogether."