An Immunogenic Non-Human Sialic Acid in Human ES Cells.
Authors: Martin Maria J. , Muotri Alysson , Gage Fred , Varki Ajit 
Published: April 2020

Abstract
The potential of human embryonic stem cells (HESC) to differentiate into any cell type or tissue makes them excellent candidates for therapy of many diseases. To be safe for transplantation, HESC-derived cells and tissues must be free of xenogenic components that could trigger an immune response in the patient. Almost all currently existing HESC cell lines have been in contact with non-human cells (a mouse embryonic fibroblast feeder layer) and/or animal products (such as fetal calf serum or “serum replacements”). These xenogenic culture conditions not only increase the risk of infection by non-human pathogens but also, as we address here, the possibility for HESC to incorporate the immunogenic non-human sialic acid N-glycolylneuraminic acid (Neu5Gc). We used flow cytometry to detect Neu5Gc on surfaces of HESC growing under such standard culture conditions and confirmed its presence by chemical analysis, showing that it represented up to 10% of the total sialic acids in HESC. In embryoid bodies (EB), the first step in HESC differentiation, the percentage of Neu5Gc ranged from 5% to 17%, despite being grown in the absence of the feeder layer and with a reduced concentration of serum. While the HESC could incorporate some Neu5Gc from the feeder layer, the major source appears to be the serum-replacement medium, which was found to be very rich in Neu5Gc (129 nmoles/mL). As many healthy humans have “natural” circulating anti-Neu5Gc antibodies (Tangvoranuntakul P et al. Proc Natl Acad Sci USA2003; 100, 12045–12050), we asked if these antibodies could recognize the HESC growing under the standard conditions and if this binding caused activation of complement. Human IgG and complement factor C3b were indeed detected on HESC cell surfaces after exposure to human sera with high level of anti-Neu5Gc antibodies. Much less IgG or C3b was detected after exposure to human sera with low level such antibodies. To reduce the Neu5Gc content of HESC and EB, we substituted the serum-replacement medium with heat inactivated type AB pooled human serum batches that were also pretested to be very low in natural anti-Neu5Gc antibodies. HESC were able to maintain an undifferentiated state when cultured in such conditions, without losing the ability to differentiate into EB. After a week in the new medium, the percentage of Neu5Gc dropped to 1.2% in the HESC and to 0.2% in the EB. Moreover, this process markedly reduced the IgG and C3b deposition caused by exposure to human sera with high levels of anti-Neu5Gc antibodies. Thus, the metabolic uptake and incorporation of Neu5Gc by HESC growing under the currently accepted culture conditions could trigger an immune response against any HESC-derived transplant, if the recipient has naturally occurring anti-Neu5Gc antibodies. We demonstrate that growth and maintenance of HESC in human serum is feasible, and markedly reduces this risk. In practice, a short-term switch to serum from the transplant recipient should be sufficient to eliminate the Neu5Gc risk. And added advantage of this approach is that one can screen for any allogeneic cytotoxic antibodies that happen to be in the transplant recipient’s serum.