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Human stem cells contain substantial amounts of the xenoantigen N‐glycolylneuraminic acid (Neu5Gc), although the levels of Neu5Gc are low or undetectable in human body fluids and most other human tissues. The lack of Neu5Gc in human tissues has been previously explained by the loss of hydroxylase activity of the human CMP‐ N ‐acetylneuraminic acid hydroxylase (CMAH) protein caused by a genetic error in the human  Cmah  gene. We thus wanted to investigate whether the human redundant  Cmah  gene could still function in stem cell‐specific processes. In this study, we show that CMAH gene expression is significantly upregulated in the adult stem cell populations studied, both of hematopoietic and mesenchymal origin, and identify CMAH as a novel stem cell marker. The CMAH content co‐occurs with higher levels of Neu5Gc within stem cells as measured by mass spectrometric profiling. It seems that despite being enzymatically inactive, human CMAH may upregulate the Neu5Gc content of cells by enhancing Neu5Gc uptake from exogenous sources. Furthermore, exposure to exogenous Neu5Gc caused rapid phosphorylation of β‐catenin in both CMAH overexpressing cells and bone marrow‐derived mesenchymal stem cells, thereby inactivating Wnt/β‐catenin signaling. The data demonstrate the first molecular evidence for xenoantigen Neu5Gc‐induced alteration of crucial stem cell‐specific signaling systems for the maintenance of self renewal. These results add further emphasis to the crucial need for completely xenofree culturing conditions for human stem cells. S TEM  C ELLS  2010;28:258–267

Human CMP‐ N ‐Acetylneuraminic Acid Hydroxylase Is a Novel Stem Cell Marker Linked to Stem Cell‐Specific Mechanisms